Hormonal control of intestinal Fc receptor gene expression and immunoglobulin transport in suckling rats.

Hormonal control of immunoglobulin (Ig) absorption and of intestinal Fc receptor mRNA expression were investigated in rats to assess its potential role in the normal postsuckling inhibition of this transport system. Corticosterone and L-thyroxine therapy caused premature inhibition of the absorption of orally administered murine monoclonal antibody and of Fc receptor mRNA expression in a dose- and time-dependent manner. Low-dose corticosterone had no effect on Fc receptor mRNA synthesis after 3 d but decreased Ig transport fivefold after 7 d. High dose corticosterone resulted in a threefold reduction in Fc receptor after 3 d, and there was almost complete inhibition (> 30-fold) of transport and of Fc receptor transcript levels after 7 d. Similarly, 7 d of high-dose thyroxine decreased both serum Ig transport and Fc receptor (> 30-fold). However, adrenalectomy did not prevent the normal post-suckling declines in Ig transport or receptor synthesis. This study demonstrates that exogenous corticosteroids and thyroxine hormone inhibit Ig transport and steady-state duodenal Fc receptor mRNA levels in suckling rats. Endogenous adrenal steroids however, do not appear to be entirely responsible for the age-dependent decline in this transport system.

Peripheral corticotropin releasing factor (CRF) and a novel CRF1 receptor agonist, stressin1-A activate CRF1 receptor expressing cholinergic and nitrergic myenteric neurons selectively in the colon of conscious rats.

Intraperitoneal (i.p.) corticotropin releasing factor (CRF) induced a CRF(1) receptor-dependent stimulation of myenteric neurons and motility in the rat proximal colon. We characterize the colonic enteric nervous system response to CRF in conscious rats. Laser capture microdissection combined with reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry in longitudinal muscle myenteric plexus whole-mount colonic preparations revealed CRF(1) receptor expression in myenteric neurons. CRF (i.p., 10 microg kg(-1)) induced Fos immunoreactivity (IR) (cells per ganglion) selectively in myenteric plexus of proximal (18.3 +/- 2.4 vs vehicle: 0.0 +/- 0.0) and distal colon (16.8 +/- 1.2 vs vehicle: 0.0 +/- 0.0), but not in that of gastric corpus, antrum, duodenum, jejunum and ileum. The selective CRF(1) agonist, stressin(1)-A (i.p., 10 microg kg(-1)) also induced Fos IR in myenteric but not in submucosal plexus of the proximal and distal colon. Fos IR induced by CRF was located in 55 +/- 1.9% and 53 +/- 5.1% of CRF(1) receptor-IR myenteric neurons and in 44 +/- 2.8% and 40 +/- 3.9% of cholinergic neurons with Dogiel type I morphology, and in 20 +/- 1.6% and 80 +/- 3.3% of nitrergic neurons in proximal and distal colon respectively. CRF and stressin(1)-A elicit defecation and diarrhoea. These data support that one mechanism through which peripherally injected CRF ligands stimulate colonic function involves a direct action on colonic cholinergic and nitrergic myenteric neurons expressing CRF(1) receptor.

CCKA and CCKB receptors are expressed in small cell lung cancer lines and mediate Ca2+ mobilization and clonal growth.

Gastrin, cholecystokinin (CCK), and CCK-related peptides comprise a hormonal family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium ([Ca2+]i). Furthermore, gastrin acts as a direct growth factor through CCKB/gastrin receptors. We report here that the expression of the mRNA coding for CCKB/gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of Ca2+ mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expression of CCKB/gastrin receptor mRNA. Accordingly, gastrin (1-100 nM) did not cause any measurable increase in [Ca2+]i. In contrast, the addition of cholecystokinin residues 26-33 (CCK-8) caused a rapid and transient increase in [Ca2+]i in this cell line. CCK-8 mobilized Ca2+ in a dose-dependent manner in the nanomolar range (half-maximal stimulatory concentration = 12 nM). Furthermore, the selective CCKA antagonist CAM-1481 inhibited the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 3 nM) in GLC19 but not in H510 cells. The selective CCKB/gastrin antagonist blocked the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 cells in a dose-dependent manner in the nanomolar range, whereas over the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but not in H510 cells. Our results show, for the first time, that CCKA receptors can mediate Ca2+ mobilization and growth in SCLC cells and that SCLC cells express two distinct functional CCK receptor subtypes.

Intracisternal antisense oligodeoxynucleotides to the thyrotropin-releasing hormone receptor blocked vagal-dependent stimulation of gastric emptying induced by acute cold in rats.

Cold exposure increases TRH gene expression in hypothalamic and raphe nuclei and results in a vagal activation of gastric function. We investigated the role of medullary TRH receptors in cold (4-6 C, 90 min)-induced stimulation of gastric motor function in fasted conscious rats using intracisternal injections of TRH receptor (TRHr) antisense oligodeoxynucleotides (100 microg twice, -48 and -24 h). The gastric emptying of a methyl-cellulose solution was assessed by the phenol red method. TRH (0.1 microg) or the somatostatin subtype 5-preferring analog, BIM-23052 (1 microg), injected intracisternally increased basal gastric emptying by 34% and 47%, respectively. TRHr antisense, which had no effect on basal emptying, blocked TRH action but did not influence that of BIM-23052. Cold exposure increased gastric emptying by 64%, and the response was inhibited by vagotomy, atropine (0.1 mg/kg, i.p.), and TRHr antisense (intracisternally). Saline or mismatched oligodeoxynucleotides, injected intracisternally under similar conditions, did not alter the enhanced gastric emptying induced by cold or intracisternal injection of TRH or BIM-23052. These results indicate that TRH receptor activation in the brain stem mediates acute cold-induced vagal cholinergic stimulation of gastric transit, and that medullary TRH may play a role in the autonomic visceral responses to acute cold.

Somatostatin 2A receptor is expressed by enteric neurons, and by interstitial cells of Cajal and enterochromaffin-like cells of the gastrointestinal tract.

Somatostatin exerts multiple effects by activating distinct G protein-coupled receptors. Here we report the cellular sites of expression of the somatostatin subtype 2A (sst2A) receptor in the rat enteric nervous system by using a C-terminus-specific, affinity-purified antiserum and immunohistochemistry. Antibody specificity was confirmed by the cell surface staining of human embryonic kidney 293 cells expressing the sst2A receptor, the lack of staining of cells expressing the somatostatin subtype 2B receptor, and the abolition of staining by preincubating the antiserum with the C-terminus peptide used for immunization, SSt2A(361-369). The SSt2A receptor antibody recognized a broad 80 kDa band on Western blots of membranes prepared from cells transfected with sst2A receptor cDNA; following receptor membrane deglycosylation, the antibody detected an additional 40 kDa band. In the enteric nervous system, the sst2A antibody primarily stained neurons of the myenteric and submucosal plexuses, and abundant fibers distributed to the muscle, mucosa, and vasculature. Immunoreactive staining was also observed in non-neuronal cells, including presumed interstitial cells of Cajal of the intestine and enterochromaffin-like cells of the stomach. Fibers expressing sst2A receptor immunoreactivity were often in close proximity to D cells of the gastric and intestinal mucosa. Colocalization of somatostatin and sst2A receptor immunoreactivities was not observed in endocrine cells nor in enteric neurons. Double-label immunohistochemistry revealed colocalization of sst2A and vasoactive intestinal peptide immunoreactivities in enteric neurons. The multiple types of cells expressing the sst2A receptor, including enteric neurons and non-neuronal structures, in addition to the relationship between somatostatin and sst2A receptor elements, provide evidence that the sst2A receptor mediates somatostatin effects in the gastrointestinal tract via neuronal and paracrine pathways.

A simple, computer-assisted assay to detect isotype-specific regulation of human immunoglobulin synthesis.

A simple, reliable, and computer-assisted assay has been developed to quantitate isotype-specific regulation of human immunoglobulin synthesis in vitro. The assay utilizes three separate human lymphoblast or myeloma cell lines, which secrete human immunoglobulins IgA, IgG, and IgE. Culture supernatants from 96-well tissue culture plates are then assayed for IgA, IgG, and IgE by a solid-phase enzyme-linked immunosorbent assay (ELISA) on a microtiter plate. Data collection and analysis is performed with the aid of computer programs designed for this assay. This assay has several advantages over other immunoglobulin regulation assays: no radioisotopes are used, thereby reducing cost and complexity; results are directly collected and quantified by computer analysis; the entire assay is completed in 3 days; reliability and reproducibility are increased by the use of established human cell lines; and co-culturing all three immunoglobulin-producing cell lines provides convenient internal controls for isotype specificity.

Cold exposure elevates thyrotropin-releasing hormone gene expression in medullary raphe nuclei: relationship with vagally mediated gastric erosions.

The stimulation of thyrotropin release by cold is associated with an increase in thyrotropin-releasing hormone gene expression in the paraventricular nucleus of the hypothalamus. Cold exposure also stimulates autonomic outflow to viscera. There is evidence that caudal raphe nuclei are involved in autonomic regulation through thyrotropin-releasing hormone projections to the dorsal vagal complex and spinal cord. To determine whether cold modulates thyrotropin-releasing hormone gene expression in the caudal raphe nuclei, the effect of cold exposure on thyrotropin-releasing hormone messenger RNA levels in the rat lower brainstem was examined by quantitative Northern blot analysis and thyrotropin-releasing hormone messenger RNA was localized by in situ hybridization. The gastric responses to cold exposure were also assessed in sham or vagotomized rats with pylorus ligation. Thyrotropin-releasing hormone messenger RNA signal was detected in the RNA extracted from the medulla and hypothalamus but not from the amygdala, periaqueductal gray or cerebellum. Cold exposure (4 degrees C) for 1 or 3 h increased thyrotropin-releasing hormone messenger RNA levels in the medulla by 77 +/- 37 and 142 +/- 39% respectively. In situ hybridization histochemistry showed that the increase in silver grain density occurred exclusively in the raphe pallidus and raphe obscurus. Exposure to cold stress for 2 h stimulated gastric acid secretion and resulted in gastric lesion formation in sham but not vagotomized rats. There are established thyrotropin-releasing hormone projections from the raphe pallidus and obscurus to the dorsal vagal complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral secretin-induced Fos expression in the rat brain is largely vagal dependent.

I.v. injection of secretin activates neurons in brain areas controlling autonomic function and emotion. Peripheral administration of secretin inhibits gastric functions through a central mechanism that is mediated by vagal dependent pathways. We investigated whether the vagus nerve is involved in i.p. injection of secretin-induced brain neuronal activation in conscious rats as monitored by Fos immunohistochemistry. Secretin (40 or 100 microg/kg, i.p., 90 min) induced a dose-related increase in the number of Fos positive neurons in the central nucleus of the amygdala (CeA), and a plateau Fos response in the area postrema (AP), nucleus tractus solitarii (NTS), locus coeruleus (LC), Barrington's nucleus (Bar), external lateral subnucleus of parabrachial nucleus (PBel) and arcuate nucleus, and at 100 microg/kg, in the dorsal motor nucleus of the vagus (DMV) compared with i.p. injection of vehicle. Double immunohistochemistry showed that secretin (40 microg/kg, i.p.) activates tyrosine hydroxylase neurons in the NTS. Subdiaphragmatic vagotomy (7 days) abolished Fos expression-induced by i.p. secretin (40 microg/kg) in the NTS, DMV, LC, Bar, PBel and CeA, while a significant rise in the AP was maintained. In contrast, s.c. capsaicin (10 days) did not influence the Fos induction in the above nuclei. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR showed that secretin receptor mRNA is expressed in the nodose ganglia and levels were higher in the right compared with the left ganglion. These results indicate that peripheral secretin activates catecholaminergic NTS neurons as well as neurons in medullary, pontine and limbic nuclei regulating autonomic functions and emotion through vagal-dependent capsaicin-resistant pathways. Secretin injected i.p. may signal to the brain by interacting with secretin receptors on vagal afferent as well as on AP neurons outside the blood-brain barrier.

Studies of regulation of gastrin synthesis and post-translational processing by molecular biology approaches.

We have examined the regulation of gene expression and post-translational processing of progastrin by starvation and feeding in rats. An oligonucleotide complementary to rat preprogastrin cDNA was used in RNA blot and hybridization analysis to measure gastrin mRNA levels. A region-specific antibody raised against the predicted amino acid sequence of the carboxyl terminal extension of progastrin was used for quantitation of progastrin peptides. The effects of starvation and of refeeding on rat antral gastrin mRNA and pro-hormone peptide levels were examined in rats starved for 48 h and after refeeding with a solid meal. Antral gastrin mRNA concentrations decreased to a plateau level (30% of the nonstarved control) after 48 h of starvation. Immunoreactive gastrin concentration decreased threefold, but progastrin processing intermediates did not decrease significantly during fasting. Following refeeding, significant increases in antral mRNA level were detected in 1 h, and peak levels were reached by 2 h (more than two times higher than starved control). There was a rapid and significant decrease in progastrin immunoreactivity within 30 min, followed by a significant increase in gastrin immunoreactivity 2 h after refeeding. These data suggest that rapid increases of blood and tissue gastrin levels in response to food may be associated with increases in both gastrin gene expression and post-translational processing of progastrin.

Regulation of gastric somatostatin gene expression.

Regulation of somatostatin gene expression was studied in the rat gastric antrum. Antral total RNA was isolated from animals during starvation and after refeeding, or under gastric neutralization by fundectomy or by omeprazole treatment. Northern blot analysis using cRNA probe synthesized from a cloned rat somatostatin cDNA demonstrated a single hybridizing band, approximately 850 nucleotides in length, which is present in the antrum. Quantitative slot blot analyses were able to detect significant changes of somatostatin mRNA levels in total RNA as low as 5 micrograms. Somatostatin mRNA levels increased significantly after 12 hours of fasting (144% of control) and remained elevated throughout the 4-day fasting period. Upon refeeding with solid food and phenylalanine, antral somatostatin returned to the prefasted level in 2 hours. Refeeding with olive oil or saline depressed somatostatin mRNA significantly within 30 to 60 minutes but did not attain the prefasted state. Fundectomy and omeprazole resulted in maximal inhibition of antral somatostatin mRNA levels by 77% and 78%, respectively. The present in vivo results indicate that somatostatin gene expression in the stomach is regulated by luminal factors that include pH and specific nutrients. Future studies based on this phenomenon can expand knowledge of the interactions between gastric endocrine cells and the gastric environment.

Regulation of cholecystokinin-mediated amylase secretion by leptin in rat pancreatic acinar tumor cell line AR42J.

Expression of the long form of the leptin receptor, the isoform that is considered to have full signaling capability, has been reported in the central nervous system and several peripheral cell types. However, only a few cell lines have been shown to express the long form of the receptor. AR42J, a cell line derived from azaserine-treated rat pancreas, is a common model for pancreatic acinar cell secretion. In this study, the presence of leptin-receptor variants and leptin action was evaluated in this cell line. Messenger RNAs for both the long and a short form of the leptin receptor were detected by reverse transcription-polymerase chain reaction (RT-PCR) in AR42J cells, and authenticity of the receptor was confirmed by DNA sequencing. Competitive binding studies demonstrated that binding of radiolabeled leptin was specific and did not cross-react with cholecystokinin (CCK). Biologic effects of leptin on amylase release and intracellular calcium mobilization were further assessed in the presence and the absence of CCK, a known pancreatic secretagogue. Although leptin alone (< or =200 ng/ml) did not affect basal amylase release, it inhibited amylase release stimulated by 1 nM CCK by 48%. Leptin alone had no significant effect on calcium mobilization. However, pretreatment of leptin (10 and 100 ng/ml) enhanced calcium responses stimulated by CCK. These data demonstrate that the rat pancreatic tumor cell line AR42J expresses a functional form of the leptin receptor that modulates the action of CCK in calcium mobilization and amylase release.

Corticotropin releasing factor in the rat colon: expression, localization and upregulation by endotoxin.

Little is known about CRF expression and regulation in the rat colon compared to the brain. We investigated CRF gene expression, cellular location, and regulation by endotoxin and corticosterone in the male rat colon at 6h after intraperitoneal (ip) injection. CRF mRNA level, detected by reverse transcription-polymerase chain reaction (RT-PCR) was 1.3-fold higher in the distal than proximal colon and 3.4-fold higher in the proximal colonic submucosa plus muscle layers than in mucosa. CRF immunoreactivity was located in the epithelia, lamina propria and crypts, and co-localized with tryptophan hydroxylase, a marker for enterochromaffin (EC) cells, and in enteric neurons. Lipopolysaccharide (LPS, 100 microg/kg, ip) increased defecation by 2.9-fold and upregulated CRF mRNA by 2.5-fold in the proximal and 1.1-fold in the distal colon while there was no change induced by corticosterone as monitored by quantitative PCR. LPS-induced increased CRF mRNA expression occurred in the submucosa plus muscle layers (1.5-fold) and the mucosa of proximal colon (0.9-fold). LPS increased significantly CRF immunoreactivity in the submucosal and myenteric plexuses of proximal and distal colon compared to saline groups. These results indicate that in rats, CRF is expressed in both proximal and distal colon and more prominently in enteric neurons of the submucosa plus muscle layers and subject to upregulation at the gene and protein levels by LPS through corticosteroid independent pathways. These data suggests that colonic CRF may be part of the local effector limb of the CRF(1) receptor mediated colonic alterations induced by acute stress.

Ontogenetic development and distribution of antibody transport and Fc receptor mRNA expression in rat intestine.

The intestine of the suckling rat has the unique capacity of absorbing immunoglobulins from maternal milk. We investigated intestinal Fc receptor mRNA expression and the absorption of orally administered antibodies to delineate the ontogeny and tissue specificity of this transport system. Duodenal expression of Fc receptor mRNA was at maximum levels between 1 and 19 days of age, but was not detectable during fetal life and in animals after weaning. Along the horizontal axis of the intestine, FcRn mRNA expression was maximum in the proximal duodenum and declined gradually in distal bowel. Similarly, absorption of orally administered antibody was low shortly after birth, but reached maximum levels at 14 days of age. By the time of weaning, antibody uptake had almost completely ceased. These data further delineate the temporal and spatial nature of the intestinal immunoglobulin transport system, and represent additional examples of how the intestinal Fc receptor is transcriptionally regulated.

Stimulation of amylase release by Orexin is mediated by Orexin 2 receptor in AR42J cells.

INTRODUCTION AND AIMS: Orexins have been demonstrated to have mainly central physiological functions, including regulation of food and water intake, sleep, and arousal. However, little is known about their direct peripheral effects, if any. As a first step toward understanding the role of Orexin in non-neuronal tissues or cells, we initiated studies to examine expression of Orexin receptors (OXR) in an established pancreatic tumor cell line AR42J. Secondly, we wanted to determine whether Orexins, in various molecular forms, are active to stimulate any known pancreatic cell functions in AR42J cells. METHODOLOGY: Reverse transcription-PCR analysis was performed to identify the presence of specific Orexin receptor subtypes. Intracellular calcium mobilization and cAMP levels were measured following stimulation by Orexin A and B peptides, their respective C-terminal decapeptide fragments, and hypocretin-2-gly (glycine-extended Orexin B). Release of alpha-amylase was measured in conditioned media after acute stimulation with the set of Orexin peptides for 30 minutes. Cell proliferation was determined by H-thymidine incorporation after 24 hours following treatment with Orexins under serum-free condition. RESULTS: RT-PCR and sequencing results showed that Orexin receptor subtype 2 (OX2R) was the main form expressed in AR42J cells. Orexins stimulated dose-dependent increases in intracellular calcium mobilization with EC50 0.05 nM for Orexin A and 0.1 nM for Orexin B but were unable to stimulate any significant cAMP accumulation or DNA synthesis even at micromolar concentrations. Both Orexin-A and -B, but not hypocretin-2-gly, also stimulated dose-dependent increases in amylase release in the AR42J cells. Orexin-A and -B carboxyl-terminal decapeptides elicited significant but much lower calcium and amylase responses. CONCLUSION: Our data demonstrate that OX2R mediates Ca -dependent amylase release in AR42J cells, suggesting that Orexins may have secretory functions in pancreatic tumor cells.

Stem cell factor alters membrane potential of purified peritoneal mast cells in culture.

The membrane potential (E(m)) was used as an indicator to evaluate the effect of stem cell factor (SCF) on the membrane integrity of peritoneal mast cells (PMCs). PMCs were harvested from the peritoneal lavage of Sprague-Dawley rats, purified more than 95% and cultured with or without the presence of SCF (2 x 10(-8) M). E(m) values were measured with conventional intracellular recording techniques. Results from day 1 to day 4 in culture were compared. Significant differences in average E(m) (aE(m)) (P < 0.01, analysis of variance) were seen on days 3 and 4 (means +/- SE in millivolts): -67.4 +/- 8.0 and -59.4 +/- 4.8 with SCF vs. -24.8 +/- 7.9 and -7.6 +/- 3.9 without SCF, respectively. Moreover, after culture with SCF for >1 wk, the aE(m) values of purified PMCs had a tendency to reach plateau values similar to that of unpurified PMCs on day 1 (at -20 mV). The morphological appearances of PMCs can be correlated with the results of aE(m) measurements. PMCs with a smooth spherical shape and highly refractive appearance, and better tolerance to electrode impalement, showed E(m) with greater negative values and lesser fluctuations. These results indicate that SCF can maintain the membrane properties and viability of purified PMCs in a long-term culture.

Peptide YY inhibition of rat gastric enterochromaffin-like cell function.

BACKGROUND & AIMS: The cellular target for peptide YY (PYY) inhibition of gastric acid secretion is unknown. The aim of this study was to determine whether PYY inhibits histamine release from isolated enterochromaffin-like (ECL) cells by stimulation of a selective Y receptor. METHODS: Isolated rat gastric ECL cells were analyzed in short-term culture for histamine release and for changes in intracellular calcium concentration using video imaging. RESULTS: Gastrin-stimulated histamine release was inhibited with a 50% inhibiting concentration of 2.10(-9) mol/L. Inhibition of histamine release and of calcium entry by PYY and [Pro34]PYY and no effect of PYY(3-36) characterizes an inhibitory Y1 receptor subtype. Reverse-transcription polymerase chain reaction of ECL cell RNA showed that the receptor was the nontruncated Y1 isoform. The inhibitory action of PYY and related peptides on gastrin-stimulated histamine release and calcium signaling was eliminated by pretreatment with 200 ng/mL pertussis toxin. Additive but not synergistic inhibitory effects of PYY and somatostatin on gastrin-stimulated histamine release were observed. CONCLUSIONS: Activation of a Y1 inhibitory receptor subtype present on the gastric ECL cell that inhibits gastrin-induced ECL cell histamine release and Ca2+ entry by activation of a Gi or G(o) class of protein may account for inhibition of gastric acid secretion by PYY released from the small intestine.

Immunolocalization of gastrin-dependent histidine decarboxylase activity in rat gastric mucosa during feeding.

The localization of histidine decarboxylase (HDC) activity in the enterochromaffin-like (ECL) cells of the oxyntic mucosa was studied during fasting and refeeding using monoclonal (CURE no. 44178) and polyclonal (CURE no. 94211) antibodies directed against the COOH terminus of HDC (HDC-CT). Changes in HDC immunostaining were correlated with mucosal HDC enzyme activity. Immunoneutralization of circulating gastrin and atropine treatment during refeeding were used to determine the relative importance of gastrin and cholinergic mechanisms in the regulation of HDC activity and immunostaining. Fasting caused a rapid reduction in the number of ECL cells immunostaining for HDC that was correlated with an almost complete loss of mucosal HDC enzyme activity. Refeeding restored both HDC immunostaining and enzyme activity within 2-4 h, and this response was inhibited by gastrin immunoneutralization but not by atropine treatment. Immunostaining was uniformly decreased and restored in the lower half of the oxyntic mucosa, which corresponds to the predominant area of ECL cells in the gastric gland. Histamine immunostaining and mucosal histamine content were not significantly changed during fasting and refeeding or by gastrin antibody and/or atropine treatment during refeeding. These findings indicate that HDC activity correlates with HDC-CT immunostaining and that both HDC activity and HDC-CT immunostaining are regulated by gastrin during refeeding.

Molecular characterization and physiological regulation of a TATA-less gene encoding chicken gastrin.

Avian gastrin is a gastric acid secretagogue, but is structurally more closely related to mammalian cholecystokinin, which is functionally distinct from gastrin. This apparently anomalous structure/activity relationship raises important issues for understanding the evolution of regulatory peptides and the mechanisms that control their expression. To clarify the possible mechanisms, we have determined the sequence of a 6.5-kb BamHI genomic DNA fragment that includes the entire avian gastrin transcriptional unit. The complete cDNA sequence, determined by anchored PCR, encoded a precursor of 105 amino acids. Conserved primary amino acid structures were limited to a decapeptide determining biological activity, and essential sites for post-translational processing. Significantly, however, the genomic regulatory regions, and introns, were unlike those of any previously reported gastrin/cholecystokinin gene. The avian gastrin gene contained no recognizable TATA-box motif, a feature unique to this gene family, but had a G+C-rich region immediately upstream of the transcription initiation site, and a Z-DNA purine-pyrimidine repeat sequence. Moreover, physiological regulation of the avian gastrin gene differed markedly from that observed in mammals, in that the important local paracrine downregulation by antral somatostatin was absent; instead, evidence for a hormonal regulation was demonstrated. The data indicate that genomic regulatory elements within regulatory peptide families such as the gastrin/cholecystokinin family, and mechanisms of physiological control, are not conserved during evolution, even though biologically important amino acid sequence information is retained.

The somatostatin receptor subtype on rat enterochromaffinlike cells.

BACKGROUND/AIMS: Gastric enterochromaffinlike (ECL) cells play an important role in peripheral regulation of acid secretion. This study investigated the somatostatin receptor subtype on ECL cells. METHODS: ECL cells were isolated from rat fundic mucosa to a purity of 90%-95% by combining enzymatic digestion, elutriation, density gradient centrifugation, and culture. RESULTS: Polymerase chain reaction performed with templates from an ECL cell complementary DNA library and primers specific to each of the five known somatostatin receptor subtypes showed that the somatostatin receptor type 2 was significantly enriched in ECL complementary DNA. Single cell videoimaging of highly purified ECL cells in culture showed that only the somatostatin receptor type 2 selective agonist, DC 32-87, inhibited the gastrin-induced calcium signal at 10(-11) mol/L. The type 3 and type 4 selective agonists, DC 25-12 and DC 32-92, and also somatostatin 14 required 100-1000 times higher concentrations (10(-8) mol/L). The somatostatin receptor type 2 analogue also inhibited gastrin-stimulated histamine release with a 50% inhibitory concentration (IC50) value of 2 x 10(-12) mol/L, whereas somatostatin 14 and the type 3 and 4 analogues showed IC50 values of 1 to 5 x 10(-9) mol/L. CONCLUSIONS: The predominant somatostatin receptors on rat gastric ECL cells are of the somatostatin receptor 2 subtype; they inhibit histamine secretion by interfering with the gastrin-induced calcium signal.