Dopaminergic and serotonergic modulation of social reward appraisal in zebrafish (Danio rerio) under circumstances of motivational conflict: Towards a screening test for anti-compulsive drug action.

Cognitive flexibility, shown to be impaired in patients presenting with compulsions, is dependent on balanced dopaminergic and serotonergic interaction. Towards the development of a zebrafish (Danio rerio) screening test for anti-compulsive drug action, we manipulated social reward appraisal under different contexts by means of dopaminergic (apomorphine) and serotonergic (escitalopram) intervention. Seven groups of zebrafish (n = 6 per group) were exposed for 24 days (1 h per day) to either control (normal tank water), apomorphine (50 or 100 µg/L), escitalopram (500 or 1000 µg/L) or a combination (A100/E500 or A100/E1000 µg/L). Contextual reward appraisal was assessed over three phases i.e. Phase 1 (contingency association), Phase 2 (dissociative testing), and Phase 3 (re-associative testing). We demonstrate that 1) sight of social conspecifics is an inadequate motivational reinforcer under circumstances of motivational conflict, 2) dopaminergic and serotonergic intervention lessens the importance of an aversive stimulus, increasing the motivational valence of social reward, 3) while serotoninergic intervention maintains reward directed behavior, high-dose dopaminergic intervention bolsters cue-directed responses and 4) high-dose escitalopram reversed apomorphine-induced behavioral inflexibility. The results reported here are supportive of current dopamine-serotonin opponency theories and confirm the zebrafish as a potentially useful species in which to investigate compulsive-like behaviors.

Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria.

Gastric acid exerts a feedback inhibition on the secretion of gastrin from antral G cells. This study examines whether gastrin gene expression is also regulated by changes in gastric pH. Achlorhydria was induced in rats by the gastric H+/K+ ATPase inhibitor, omeprazole (100 mumol/kg). This resulted in fourfold increases in both serum gastrin (within 2 h) and gastrin mRNA levels (after 24 h). Antral somatostatin D cells probably act as chemoreceptors for gastric acid to mediate a paracrine inhibition on gastrin secretion from adjacent G cells. Omeprazole-induced achlorhydria reduced D-cell activity as shown by a threefold decrease in antral somatostatin mRNA levels that began after 24 h. Exogenous administration of the somatostatin analogue SMS 201-995 (10 micrograms/kg) prevented both the hypergastrinemia and the increase in gastrin mRNA levels caused by omeprazole-induced achlorhydria. Exogenous somatostatin, however, did not influence the decrease in antral somatostatin mRNA levels seen with achlorhydria. These data, therefore, support the hypothesis that antral D cells act as chemoreceptors for changes in gastric pH, and modulates somatostatin secretion and synthesis to mediate a paracrine inhibition on gastrin gene expression in adjacent G cells.

Pancreatic gastrin stimulates islet differentiation of transforming growth factor alpha-induced ductular precursor cells.

Gastrin is transiently expressed in fetal islets during a critical period of their development from protodifferentiated islet precursors in fetal pancreatic ducts. To examine the possible role of gastrin as an islet cell growth factor, postnatal islet growth was studied in transgenic mice which overexpress gastrin and TGF alpha in their pancreas. Overexpression of a TGF alpha transgene causes metaplastic ductules containing numerous insulin expressing cells that resemble protodifferentiated precursors of the fetal pancreas. However, islet mass of the TGF alpha transgenic mice was not increased. Pancreatic overexpression of gastrin from a chimeric insulin/gastrin transgene transcribed from the insulin promoter markedly decreased the TGF alpha-stimulated increase in pancreatic duct mass. Furthermore, pancreatic coexpression of both gastrin and TGF alpha significantly increased islet mass in mice expressing both transgenes. These findings indicate that TGF alpha and gastrin can act synergistically to stimulate islet growth, although neither peptide alone is sufficient. Islet growth may possibly be stimulated through gastrin promoting the differentiation of insulin-positive cells in the TGF alpha-induced metaplastic ducts. This transgenic study suggests that islet neogenesis can be reactivated in the ductular epithelium of the adult pancreas by local expression of two growth factors, gastrin and TGF alpha.

A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter.

Epidermal growth factor (EGF) and transforming growth factor alpha are important determinants of mucosal integrity in the gastrointestinal tract, and they act both directly and indirectly to prevent ulceration in the stomach. Consistent with this physiological role, EGF stimulates transcription of gastrin, a peptide hormone which regulates gastric acid secretion and mucosal growth. EGF stimulation of gastrin transcription is mediated by a GC-rich gastrin EGF response element (gERE) (GGGGCGGGGTGGGGGG) which lies between -54 and -68 in the human gastrin promoter. The gERE sequence also confers weaker responsiveness to phorbol ester stimulation. The gERE sequence differs from previously described EGF response elements. The gERE DNA sequence specifically interacts with a GH4 DNA-binding protein distinct from previously described transcription factors (Egr-1 and AP2) which bind GC-rich sequences and mediate transcriptional activation by growth factors. Furthermore, the gERE element does not bind the Sp1 transcription factor even though the gERE sequence contains a high-affinity Sp1-binding site (GGCGGG).

Identification of a cis-regulatory element mediating somatostatin inhibition of epidermal growth factor-stimulated gastrin gene transcription.

Antral gastrin secretion and gene expression is inhibited by the paracrine release of somatostatin from antral D cells. Transforming growth factor-alpha and epidermal growth factor (EGF) stimulate gastrin reporter gene constructs when transfected into pituitary GH4 cells. Somatostatin inhibits EGF stimulation of gastrin gene expression, which is in part mediated at the level of transcriptional regulation as somatostatin inhibits EGF stimulation of gastrin reporter gene constructs. Somatostatin inhibition was abolished by pertussis toxin, indicating somatostatin inhibits transcription through the inhibitory G protein Gi. Somatostatin inhibition was unaffected by vanadate and okadaic acid, implying this inhibitory pathway is mediated neither through phosphotyrosine phosphatases nor serine/threonine phosphatases, respectively. Gastrin reporter genes containing 82 base pairs of the 5'-flanking DNA were sufficient to confer both EGF responsiveness and inhibition by somatostatin in GH4 cells. However, transcription of a gastrin reporter gene construct containing only the EGF response element (GGGGCGGGGTGGGGGG), located at -68 to -53, was stimulated by EGF but was not inhibited by somatostatin. Thus, somatostatin inhibits EGF-stimulated gastrin gene transcription by a mechanism other than by interfering with cell signals elicited by the EGF receptor. Since the 82 GASCAT is inhibited by somatostatin, this result also implies that sequences adjacent to the EGF response element contain a cis-regulatory element mediating transcriptional inhibition by somatostatin. This cis-element was located using gastrin reporter genes comprising sequential segments of the human gastrin promoter sequence from the transcriptional start site to -82 in the 5'-flanking DNA. Gastrin oligonucleotide constructs lacking the D oligonucleotide (gatcCATATGGCAGGGTA), located at -82 to -69 in the 5'-flanking DNA, were not inhibited by somatostatin, indicating that a somatostatin inhibitory cis-element is located between -82 and -69 in the 5'-flanking DNA of the human gastrin promoter.

Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid.

Gastrin biosynthesis involves a complex series of posttranslational modifications; their elucidation requires a knowledge of the structure of the gastrin precursor. The complete structure of rat preprogastrin was deduced from the nucleotide sequence of a full length cDNA clone isolated from a rat antral cDNA library. Northern blot hybridization analysis of rat antral RNA together with human antral RNA, reveals a single mRNA species of approximately 670 bases. Comparison of this sequence with those of porcine and human gastrin reveals extensive (73%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The rat sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment; and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. Unlike the human and porcine sequences, rat preprogastrin contains a 9 amino acid carboxy-terminal extension peptide (-Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn) which is homologous to the midportion of gastrin 17 including the site of tyrosine sulfation.

Activation of gastrin gene transcription in islet cells by a RAP1-like cis-acting promoter element.

Gastrin transcription in islet cells is activated by a cis-regulatory sequence containing a binding site for the yeast transcription factor RAP1. The DNA-protein interactions between RAP1 protein and the gastrin DNA element determined by methylation interference assays are identical to those of RAP1 and yeast genes. Point mutations in the gastrin RAP1 binding site, which abolished RAP1 binding, decreased transcriptional activation by this sequence. Islet cells revealed a DNA binding protein with RAP1-like binding specificity. These findings support the conclusion that gastrin transcription is activated in mammalian cells by a RAP1-like transcription factor.

Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease: toxins, mediators, and modulators of gene expression.

SUMMARY: : There is a growing body of both experimental and clinical evidence to suggest that chronic gut inflammation is associated with enhanced production of reactive metabolites of oxygen (e.g., superoxide, hydrogen peroxide) and nitrogen (e.g., nitric oxide). Pharmacologic intervention studies suggest that some of the tissue injury and dysfunction as well as the inflammatory process itself are mediated directly or indirectly by these oxidants and free radicals. Historically, these reactive species have been thought to promote inflammatory tissue injury via their ability to oxidize and degrade essential cellular constituents. However, more recent work suggests that oxygen-and nitrogen-derived metabolites may mediate gut pathobiology in more subtle ways. For example, nontoxic levels of superoxide- and/or nitric oxide-derived oxidants and free radicals may act as pro-inflammatory signaling agents as well as activate certain transcription factors (e.g., nuclear factor κB, activation protein [AP]-1) that are known to up-regulate the expression of a variety of different genes that are important in the inflammatory response. These data suggest that the sustained overproduction of these reactive species in the chronically inflamed gut may contribute to the pathophysiology of IBD by enhancing the production of toxins, mediators, and modulators of gene expression.

Complete sulfation of jejunal gastrin in the human fetus.

The degree of tyrosine-O-sulfation and the ratio between large (gastrin-34 and component I) and small (gastrin-17 and -14) molecular forms of gastrin were studied in extracts of human fetal (n = 14) and adult (n = 9) antrum, duodenum, jejunum and pancreas. Boiled water extracts were applied to gel- and ion-exchange chromatography before and after treatment with trypsin and arylsulfatase. The fractions were monitored with sequence-specific radioimmunoassays that distinguish sulfated from non-sulfated gastrins. In antrum and duodenum about half the gastrins were sulfated at all stages of development. In the fetal jejunum gastrin occurred in sulfated form only while in the adult 72% (range, 64-88%) of the jejunal gastrins were sulfated. The larger molecular forms of gastrin predominated in the fetal compared with the adult antrum. In duodenum and jejunum, however, the ratio between small and large forms was the same in fetus and adult. Gastrin was undetectable in both fetal and adult pancreas. The results show that the degree of sulfation of gastrin varies substantially in the different parts of the gut at different stages of development. The differences may have functional significance, since sulfation increases the pancreozyminic and cholecystokinetic potency of gastrin.

Effects of the Areca nut constituents arecaidine and guvacine on the action of GABA in the cat central nervous system.

Arecaidine and guvacine, constituents of the nut of Areca catechu, inhibited the uptake of GABA and beta-alanine, but not that of glycine, by slices of cat spinal cord. In cats anesthetised with pentobarbitone, electrophoretic arecaidine enhanced the inhibitory actions of GABA and beta-alanine, but not those of glycine or taurine, on the firing of spinal neurones. Similarly, electrophoretic guvacine enhanced the inhibition of spinal neurones by GABA but not that by glycine. The uptake of GABA by slices of cat cerebellum was inhibited by arecaidine, and the effect of electrophoretic GABA on the firing of cerebellar Purkinje cells was enhanced by electrophoretic arecaidine. When administered intravenously arecaidine failed to affect synaptic inhibitions considered to be mediated by GABA. Intravenous arecaidine had no effect on either spinal prolonged (presynaptic) inhibition (20mg/kg), dorsal root potentials (20mg/kg) or basket cell inhibition of Purkinje cells (250 mg/kg), although topical arecaidine (6.6-10 x 10(-3) M) blocked this latter inhibition. Large doses of arecaidine (1 g/kg subcutaneous) marginally reduced the lethal effects of bicuculline in mice but appeared to have little or no anticonvulsant activity.

The post-natal development of cholecystokinin-like activity in the brain and small intestine of the rat.

1. The post-natal development of cholecystokinin (CCK)-like activity was studied in the brain and small intestine of the rat. CCK-like biological activity was measured in extracts of these tissues by an in vitro rabbit gall-bladder bioassay. 2. Immediately after birth, the brain contained very little CCK-like activity whereas the proximal small intestine contained significant concentrations of CCK-like activity. The concentration of CCK-like activity in the brain increased rapidly during the third post-natal week and reached adult values by the end of the fourth week. The development of CCK-like activity in the proximal small intestine differed from that seen in the brain. The concentration of CCK-like activity increased during the first post-natal week. After this time, however, the concentration decreased and the adult values, therefore, were lower than those found immediately after birth. This decrease in concentration resulted from failure of the total content of CCK-like activity to increase despite rapid growth of the intestine. 3. The composition of CCK-like activity in neonatal extracts was determined by gel filtration chromatography with Sephadex G50. Extracts of neonatal brain and intestine contained more than one molecular form of CCK-like activity in contrast to the single peak of activity found in adult extracts. In the developing intestine smaller molecular forms were found in addition to the single larger form found in the adult and in the neonatal brain larger molecular forms were found in addition to the CCK octapeptide found in the adult.

The release of rat intestinal cholecystokinin after oral trypsin inhibitor measured by bio-assay.

The distribution, molecular form and release of cholecystokinin (CCK)-like activity in extracts of rat small intestine was studied with an in vitro gall-bladder bio-assay. In contrast to the reported heterogeneity of CCK-like immunoreactivity in the intestine, only a single molecular form of CCK-like activity was detected using the bio-assay. 2. The CCK-like activity eluted from Sephadex G50 with a Kav of 0.69, after the triacontriapeptide of cholecystokinin (CCK33) and before cholecystokinin octapeptide 2500, may represent the 22 amino acid peptide of CCK (CCK22). The bio-assay peak of CCK-like activity had pancreozymin activity and CCK/gastrin C terminal immunoreactivity. The CCK-like activity weas readily extracted from the small intestine at neutral pH, but subsequent treatment with cold 0.5 M-acetic acid extracted further CCK-like activity of the same molecular form as that recovered under neutral conditions. 3. The bio-assay detected no CCK-like activity, nor was pancreozymin-like activity found in fractions corresponding to CCK33 or CCK8 after Sephadex G50 chromatography of rat intestinal extracts. 4. Oral trypsin inhibitor was a potent stimulus for the release of CCK-like activity from the upper small intestine of the rat. After oral trypsin inhibitor release, CCK-like activity was rapidly resynthesized.

Function and regulation of gastrin in transgenic mice: a review.

The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.

The movement of an unemulsified oil test meal and aqueous- and oil-phase markers through the intestine of normal and bile-diverted rats.

An unemulsified oil test meal containing aqueous- and oil-phase markers (PEG and 3-H labelled triether) was fed to control and bile fustula rats. PEG moved ahead of the lipid phase in all groups, and was of limited value as a marker for the test meal. Triether was an excellent marker for studies of gastric emptying. An unexpectedly high correlation was seen between the triether and the test meal and its digestion products in the intestine. The results suggest that triether is a valid marker for following the movement of lipid through the bowel under the conditions of this study. Triether/fat ratios indicated that fat absorption occurred largely from the upper small bowel in control animals and from the lower small bowel in the absence of bile. Bile diversion resulted in more rapid gastric emptying initially, but very slow gastric emptying of the last part of the oil test meal. Intestinal transit of test meal or either marker was not significantly affected by bile diversion. No evidence for a direct effect of bile diversion on gastrointestinal motility was found.

The influence of starvation on intestinal cholecystokinin-like activity and pancreatic growth.

1. This study examines the influence of starvation on intestinal CCK content and pancreatic growth. Intestinal CCK content was determined by measuring the CCK-like activity using an in vitro gall-bladder bio-assay. Starvation for up to 72 hr causes a parallel fall in intestinal CCK content and pancreatic DNA synthesis. Since there was no significant decrease in liver DNA synthesis, the effect of starvation was probably not simply a consequence of malnutrition. Furthermore there was little effect of starvation on pancreatic protein and DNA content, suggesting that pancreatic cell turnover is particularly sensitive to changes in dietary stimulation.2. With refeeding after starvation CCK-like activity in intestinal extracts gradually increased, approaching non-fasting levels 72 hr after refeeding. Pancreatic DNA synthesis also returned to non-fasting levels after feeding but this rose faster than the intestinal CCK content.3. Pentagastrin treatment prevented the atrophy of both the pancreas and the gastrointestinal tract with starvation without influencing the fall in intestinal CCK-like activity. This suggests that the control of CCK-containing cells is different from that of the surrounding intestinal parenchyma.4. The effect of starvation was also studied in antrectomized rats. Antrectomy alone did not reduce pancreatic DNA synthesis although DNA synthesis of the small intestine was significantly reduced. When antrectomized rats were starved pancreatic DNA synthesis fell to the same degree as was found in unoperated animals. The pancreatic atrophy was also accompanied by a drop in intestinal CCK content. Starvation of antrectomized rats, however, did not further depress the already greatly reduced plasma gastrin concentration.

Stimulation of pancreatic secretion and growth in the rat after feeding cholestyramine.

The influence of luminal bile salts on pancreatic growth in rats was studied by feeding cholestyramine, an anion-exchange resin that binds luminal bile salts, and by chronic bile diversion. Cholestyramine given in food stimulated pancreatic growth, increasing both its size and deoxyribonucleic acid content. A dose of 0.5% was ineffective, but feeding 2%, 6%, and 10% cholestyramine (wt/wt) gave the same degree of pancreatic enlargement. Cholestyramine feeding also augmented the stimulation of pancreatic growth as seen after feeding raw soybean flour containing active trypsin inhibitor. Feeding cholestyramine increased both the basal pancreatic secretion and the secretory response to exogenous cholecystokinin. If they were fasted overnight, the pancreatic enzyme content of cholestyramine-fed rats was also increased with an equal increase in both trypsin and lipase activity. However, enzyme content after cholestyramine feeding depended on the duration of the feeding and whether or not the animals were fasted before killing. Chronic bile diversion caused pancreatic growth similar to that seen in cholestyramine-fed animals. It is suggested that the pancreatic enlargement seen after cholestyramine feeding and bile diversion is an adaption to increased secretion similar to that seen after feeding soybean trypsin inhibitor.

Gastrin gene expression and regulation in rat islet cell lines.

Gastrin gene expression was observed in two permanent rat insulinoma (RIN) cell lines derived from a rat insulinoma. Gastrin expression was selective; highest expression was seen in a cell line which did not express other islet cell hormones. Gastrin mRNA transcription initiated from the same promoter as antral gastrin mRNA. DNA transfection studies with a gastrin chloramphenicol acetyltransferase chimeric gene showed higher expression in gastrin-expressing RIN cells than non-gastrin-expressing islet cells. This implies that gastrin-expressing RIN cells selectively express a trans-acting transcriptional activator which binds to cis-acting regulatory sequences within the 5'-flanking DNA sequence and first exon of the gastrin gene. The gastrin peptide precursor synthesized in these RIN cell lines is subject to the same repertoire of posttranslational modifications within the cell's secretory apparatus (endoproteolytic cleavage, tyrosine sulfation, and C-terminal amidation) as seen in antral G cells. Gastrin mRNA levels in these RIN cells were selectively increased by increasing the extracellular calcium concentration. Membrane depolarization also stimulated gastrin mRNA levels, probably through activation of voltage-sensitive calcium channels. Thus, these gastrin-expressing RIN cell lines provide permanent cell lines useful in analyzing the cellular regulation of gastrin gene expression.