三种内分泌细胞在极北鲵幼体胃内的发育

采用免疫组织化学亲和素-生物素复合物(ABC)法研究极北鲵(Salamandrella keyserlingii)41至46期共6个时期幼体胃内5-羟色胺(5-HT)细胞、生长抑素(SS)细胞和胃泌素(GAS)细胞的分布位置及形态,5-羟色胺细胞和胃泌素细胞最先在41期幼体被检测出来,而生长抑素细胞发育略晚,在42期幼体才被检测到。文中采用Duncan多重比较的方法,对胃内同一种内分泌细胞不同发育时期的分布密度进行统计学分析,5-羟色胺细胞在41期分布最少,密度为1.2±0.51,46期分布最多,密度为2.3±0.91,随着胃的发育,5-羟色胺细胞密度呈递增的变化趋势;胃泌素细胞在42期分布最多,密度为2.1±0.99,46期分布次之,密度1.7±0.49,44期分布最少(密度1.0±0.00),从42期到46期呈先减后增的变化趋势;而生长抑素细胞在43期密度(2.6±0.99)高于其他各期,且差异显著(P0.05)。三种内分泌细胞最先均出现在胃上皮细胞之间,随着胃的不断发育,除了在上皮细胞之间分布以外,在腺泡上皮之间也有分布。不同内分泌细胞随着发育时间的不同,细胞形态也表现不同,如5-羟色胺细胞由最开始的椭圆形到后期的锥体形,胃泌素细胞由最先出现的锥体形到椭圆形再到锥体形,生长抑素细胞由锥体形到椭圆形。上述三种内分泌细胞的分布及形态学特点可能与极北鲵的生长发育以及胃的功能相适应。     

Metal ion binding affinities of gastrin and CCK in membrane mimetic environments

The fully active gastrin and CCK analogues [Nle¹⁵]-gastrin- 17 and [Nle, Thr]-CCK-9 were analysed for their Ca²⁺ and Tb³⁺ affinities in various membrane mimetic conditions. In TFE both gastrin and CCK exhibited high affinities for calcium and terbium. At saturation level identical metal ion/peptide ratios were determined with Ca²⁺ and Tb³⁺, i.e. R = 3 for gastrin and R = 1 for CCK, confirming the very similar coordination properties of the two metal ions. The conformational effects of both metal ions were found to be very similar with a disordering effect in the case of gastrin and a conformational transition to β-turn type structure in the case of CCK. In order to mimic more properly physiological conditions, similar experiments were performed in the prsence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of mmolar Ca²⁺ concentrations. Induced lipid interaction via N-terminal lipodervatization of gastrin and CCK allowed to translocate quantitatively the two hormones into phospholipid bilayers and to examine the effect of extravesicular Ca²⁺ on the conformation of the peptide headgroups and on their display at the water/lipid interphase. The CCK moiety of the lipo-CCK inserted into phospholipid bilayers interacts with the lipid phase and addition of Ca²⁺ enhances the clustering of the peptide headgroups in a more β-sheet type conformation. Conversely, insertion of lipo-gastrin into the bilayers leads to full exposure of the gastrin headgroup to the bulk water in predominantly random coil structure. Again Ca²⁺ provokes aggregation. As the lipo-peptide/phospholipid system still represents only an artificial model, it remains hazardous to derive a biological relevance from these data. The significantly higher affinity of lanthanide ions than Ca²⁺ for the peptides could well play a role in the inhibibitory activity of lanthanum on the signal transduction of the CCK family of hormones.     

Localization of carboxyl groups in gastric mucosa as possible sites of gastrin

Summary Human and pig gastrins contain a sequence of five consecutive glutamic acid residues. An attempt was made to localize gastrin using methods known or assumed to operate on a carboxyl mechanism. General methods for acidic groups were combined with selective blocking (methylation) and unblocking (saponification) methods to increase COOH specificity. Epithelial cells with weakly metachromatic granules could be identified in untreated sections stained with toluidine blue (pH 5). After prolonged methylation and saponification, the same and previously obscured cells were moderately to intensely metachromatic, this residual basophilia attributable to weak COOH groups. Specifically marked metachromatic cells were iron-positive after colloidal iron staining, but were delineated easily only after methylation-saponification. Metachromatic cells were also clearly demonstrated by the carboxyl method of Barrnett and Seligman and by silver impregnation (pH 5). The granular metachromatic cell demonstrated by these methods contains significant amounts of a weakly acidic component which the Barrnett-Seligman reaction indicates to be glutamic acid. Comparable staining results were obtained with gastrin producing Zollinger-Ellison islet cell adenomas. It is postulated that the COOH-rich substance is gastrin or gastrin precursor and that the metachromatic cell is responsible for its production.Supported by General Research Support Grant No 5 S01 FR05411-06.     

Gastrin releasing peptide reduces meal size in rats

The satiety-eliciting effect of gastrin-releasing peptide (GRP), a putative mammalian counterpart of bombesin (BBS), was examined in mildly food-deprived rats. Intraperitoneal injections of GRP resulted in a significant decrease of 30-minute food intake at 2, 4, 8 and 16 μg/kg, while 1 μg/kg had no reliable effect. Intraperitoneal GRP at 4 and 8 μg/kg did not suppress 30-minute water consumption by thirsty rats. When the dose-effect curves of GRP and BBS are compared on a molar scale, GRP is approximately 30% less potent than BBS in suppressing food intake. The two dose-effect curves are similar in shape and their regression lines have parallel slopes. These data lend further support to the hypothesis that GRP is a mammalian counterpart of BBS and strengthen the argument that they may function as endogenous satiety factors.     

By modulating α2β1 integrin signalling,gastrin increases adhesion oF AGS-GR gastric cancer cells

Peritoneal metastasis is a major cause of recurrence of gastric cancer and integrins are key molecules involved in gastric cancer cells attachment to the peritoneum. The peptide hormone, gastrin, initially identified for its role in gastric acid secretion is also a growth factor for gastric mucosa. Gastrin has also been shown to contribute to gastric cancers progression. Here, we provide the first evidence that gastrin increases the adhesion of gastric cancer cells. Gastrin treatment induces the expression of α2 integrin subunit through a mechanism that involves the ERK pathway. We also observed in response to gastrin an increase in the amount of α2 integrin associated with β1subunit. In addition, gastrin-stimulated cell adhesion was blocked with an anti-α2β1 integrin neutralizing antibody. We also show that gastrin activates the integrin pathway via the phosphorylation of β1 integrin by a Src family kinase. This mechanism may contribute to the enhancement of cell adhesion observed in response to gastrin since we found an inhibition of gastrin-mediated cell adhesion when cells were treated with a Src inhibitor. By regulating one of the key step of the metastatic process gastrin might contribute to increase the aggressive behaviour of human gastric tumours.     

Disulfide content of reduced hen egg white and human milk lysozymes during the folding process

In order to obtain a better understanding of the possible influence of the primary sequence of a protein on its folding pathway, renaturation of reduced human milk lysozyme was compared to that of reduced hen egg white lysozyme. Following disulfide bond formation, under identical conditions, similar products were found during the folding of both lysozymes, but the kinetics of appearance and disappearance of these intermediates as well as the appearance of the native conformation were different.     

Effect of synthetic porcine gastrin-releasing peptide on plasma levels of immunoreactive cholecystokinin pancreatic polypeptide and gastrin in dogs

This study was conducted to determine if synthetic porcine gastrin-releasing peptide (GRP) stimulates the release of immunoreactive cholecystokinin (CCK), pancreatic polypeptide (PP) and gastrin in dogs. Three doses (0.01, 0.1 and 0.5 μg/kg-hr) of synthetic porcine GRP were administered intravenously to six conscious dogs. Synthetic procine GRP stimulated the release of each hormone in a dose-related manner. The effect of GRP on the response of gastrin was greater than its effect on CCK and PP responses. This study indicates that the biological action of synthetic porcine GRP is similar to the bombesin, an amphibian peptide shown previously to stimulate the release of gastrointestinal peptides.     

The effect of fixation conditions on the ultrastructural appearance of gastrin cell granules in the rat gastric pyloric antrum

The ultrastructural appearance of gastrin cell (G cell) granules was studied after different fixation procedures. When the pH of prefixation was varied there was greater preservation of the electron density of granule cores after acidic (pH 5.0 and 6.0) than after neutral or alkaline (pH 7.0 and 8.0) prefixation. Increasing duration of prefixation at pH 7.3 resulted in progressive loss of electron density of the granule core with swelling and occasional rupture of the limiting membrane. In tissues where most granules had been rendered electron lucent by fixation, those granules remaining dense cored were preferentially located close to the Golgi zone. These findings indicate that the electron density of G cell granules is profoundly affected by conditions of fixation, and that immature granules are more resistant to loss of core density than mature granules. They also suggest that the gastrin granule in vivo, like other polypeptide granules, may have a "solid", osmotically inactive core.     

Characteristics of gastrin controlled ECL cell specific gene expression

BACKGROUND: The ECL cells are histamine-producing endocrine cells in the oxyntic mucosa that synthesize and secrete proteins and peptides. They are the primary target for gastrin and mediate the control of gastrin on acid secretion and oxyntic mucosal growth. Knowledge of the molecular biology of the ECL cell is therefore important for understanding gastric physiology. Accordingly, we wanted to identify genes that are characteristically expressed in the ECL cells and controlled by gastrin. METHODS: Using Affymetrix GeneChips, RNA expression profiles were generated from ECL cells isolated by counterflow elutriation from hyper- or hypogastrinemic rats. Contamination from non-endocrine cells was eliminated by subtraction of the expression profiles of the fundic and antral mucosa. RESULTS: The expression of 365 genes was ECL cell characteristic. Gastrin was found to control the expression of 120 which could be divided into two major groups depending on the known or anticipated biological function of the encoded protein: genes encoding proteins involved in the secretory process and genes encoding proteins needed to generate energy for secretion. Interestingly, gastrin stimulation also increased ECL cells expression of anti-apoptotic genes. CONCLUSION: The ECL cell specific expression profile is reminiscent of that of neurons and other endocrine cells exhibiting high expression of genes encoding proteins involved in the synthesis, storage and secretion of neuropeptides or peptide hormones. Gastrin regulated the expression of one third of these genes and is thus involved in the control of secretion from the ECL cells.     

Nonsulfated cholecystokinins in the small intestine of pigs and rats

Cholecystokinin (CCK) is a gut hormone that acts via two receptors. The CCK_A-receptor requires the tyrosyl residue in the C-terminal bioactive site of CCK to be O-sulfated, whereas, the CCK_B-receptor binds irrespective of sulfation. Consequently, unsulfated CCK peptides – if present – may constitute a hormone system that acts only through the CCK_B-receptor. Therefore, we have now examined whether, CCK peptides occur in nonsulfated form in the small intestine of pigs and rats. The concentrations of sulfated and nonsulfated CCK were measured by RIAs, one specific for sulfated CCKs and a new two-step assay specific for nonsulfated CCK. For further characterization, the intestinal extracts were subjected to size- and ion exchange-chromatography.The intestinal concentrations of sulfated and nonsulfated CCK were highest in the duodenum and the proximal part of jejunum both in the pig and the rat. The porcine duodenal mucosa contained 193 ± 84 pmol/g sulfated CCK and 31 ± 10 pmol/g nonsulfated CCK, and the upper rat intestine 70 ± 19 pmol/g and 8 ± 2 pmol/g, respectively. The degree of sulfation correlated with the endoproteolytic proCCK processing. Thus, 38% of porcine CCK-58 was unsulfated, whereas, only 12% of CCK-8 was unsulfated.The results show that a substantial part of intestinal CCK peptides in rats and pigs are not sulfated, and that the longer peptides (CCK-58 and CCK-33) are less sulfated than the shorter (CCK-22 and CCK-8). Hence, the results demonstrate that proCCK in the gut is processed both to sulfated and unsulfated α-amidated peptides of which the latter are assumed to act via the CCK_B-receptor.