Glycine-extended gastrin regulates HEK cell growth.

Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH(2)) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH(2), glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH(2) and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH(2) can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH(2) and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for (125)I-labeled Leu(15)-G17-NH(2) and (125)I-Leu(15)-G(2-17)-Gly. Both G-NH(2) and G-Gly induced a dose-dependent increase in [(3)H]thymidine incorporation, and both peptides together significantly increased [(3)H]thymidine incorporation above the level of either peptide alone. G-NH(2) and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH(2) and G-Gly lead to a significant reduction in [(3)H]thymidine incorporation. G-NH(2) but not G-Gly increased intracellular Ca(2+) concentration. We conclude that G-NH(2) and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.

Low- versus high-dose azithromycin triple therapy for Helicobacter pylori infection.

BACKGROUND: We report a clinical trial which evaluated the effectiveness of triple therapy containing low- and high-dose azithromycin to treat Helicobacter pylori infection. METHODS: From March 1997 to March 1998, patients infected with H. pylori were assigned to receive either: Treatment 1: ranitidine bismuth citrate (RBC) (400 mg b.d.) and amoxycillin (1 g b.d.) for 10 days with azithromycin 500 mg o.m. for 3 days: or Treatment 2: RBC and amoxycillin for 10 days with azithromycin 1 g o.m. for 3 days. H. pylori eradication was established by a urea breath test at least 4 weeks after therapy. Side-effects and compliance were assessed using a diary. RESULTS: Sixty-eight patients were enrolled. Fifty-seven per cent of patients were treated for active peptic ulcer disease or a history of peptic ulcer disease. Treatment 1 cured H. pylori in 44% and 44% by per protocol and intention-to-treat analysis, respectively. The corresponding eradication rates for Treatment 2 were 79% and 75%. Two patients taking Treatment 2 dropped out of the study because of side-effects. CONCLUSIONS: With RBC and amoxycillin for 10 days, azithromycin at a dose of 1 g/day for 3 days was significantly better at curing H. pylori infection than azithromycin 500 mg/day for 3 days.

Bismuth subsalicylate instead of metronidazole with lansoprazole and clarithromycin for Helicobacter pylori infection: a randomized trial.

OBJECTIVE: We evaluated the efficacy of lansoprazole, clarithromycin, and metronidazole (LCM) administered twice daily for 7 days. Because there is growing concern about the development of metronidazole-resistant H. pylori (HP) strains, we also tested a novel regimen consisting of lansoprazole, clarithromycin, and bismuth subsalicylate (LCB). METHODS: Patients with active HP infection and peptic ulcer, a history of peptic ulcer, or nonulcer dyspepsia were randomized to either lansoprazole 30 mg b.i.d., clarithromycin 500 mg b.i.d., and metronidazole 500 mg b.i.d. or lansoprazole 30 mg b.i.d., clarithromycin 500 mg b.i.d., and bismuth subsalicylate 524 mg b.i.d. (LCB) for 7 days. Compliance and side effects were recorded by using a diary. RESULTS: "Per protocol" eradication with LCM was achieved in 41 of 47 (87%). By using "intention to treat" analysis, LCM eradicated HP infection in 43 of 53 patients (81%). By using "per protocol" analysis, LCB eradicated HP infection in 40 of 47 patients (85%). On an "intention to treat" basis, LCB led to HP eradication in 42 of 52 (81%). The most common significant side effects observed with LCM were altered taste (39%) and abdominal pain (19%). With LCB, the most common significant side effects were altered taste (23%) and dark stools (23%). CONCLUSIONS: LCB for 7 days was as effective in eradicating HP infection as a 7-day course of LCM. Further studies evaluating the role of bismuth compounds in proton-pump inhibitor based triple therapy are warranted. Such therapy may have particular importance in areas where high metronidazole resistance is a concern.

Lansoprazole and ranitidine affect the accuracy of the 14C-urea breath test by a pH-dependent mechanism.

OBJECTIVES: To determine the effect of lansoprazole and high dose ranitidine on the accuracy of the 14C-urea breath test (UBT). Using intragastric pH recordings, we correlated the effect of these agents on the UBT with their potency of gastric acid suppression. METHODS: Patients with active Helicobacter pylori infection underwent a baseline UBT before receiving 14 days of lansoprazole (30 mg/day) or ranitidine (300 mg b.i.d.). During therapy, patients were asked to undergo 24-h intragastric pH monitoring. Repeat breath testing was performed 1 day after completion of the study drugs. If the UBT was equivocal or negative (14CO2 excretion was < 200 dpm), further UBTs were completed until the 14CO2 excretion was > 200 dpm. RESULTS: Thirteen patients received lansoprazole. Eight of thirteen patients developed a negative or equivocal UBT. All patients had 14CO2 excretion > 200 dpm 5 days after the cessation of lansoprazole. Eleven patients received ranitidine. Ranitidine led to equivocal or false negative UBTs in 2 of 11 cases. This effect resolved within 5 days of stopping ranitidine. Intragastric pH recordings revealed that the patients who experienced the most profound gastric acid suppression were those that developed equivocal or false negative UBTs. CONCLUSIONS: Lansoprazole significantly affected the accuracy of the UBT, causing equivocal or false negative results in 61%. High dose ranitidine affected the breath test in only 18%. The ability of these drugs to suppress gastric acid secretion predicted those patients who developed equivocal or false-negative UBTs. The effect on the accuracy of the UBT resolved within 5 days of drug cessation.

Effect of transforming growth factor alpha and interleukin 8 on somatostatin release from canine fundic D cells.

BACKGROUND & AIMS: Helicobacter pylori infection in patients who have peptic ulcer disease is associated with altered regulation of gastric secretion, hypergastrinemia, and diminished somatostatin expression in gastric mucosa. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 are the predominant cytokines produced in the gastric mucosa of patients with H. pylori infection. The aim of this study was to examine whether IL-8 and TNF-alpha could regulate somatostatin release from isolated canine gastric D cells. METHODS: Canine gastric D cells were isolated from fundic mucosa and enriched by centrifugal elutriation. Secretagogue-stimulated somatostatin release was measured by radioimmunoassay. RESULTS: TNF-alpha dose dependently increased somatostatin release after 2 hours of treatment. The stimulatory effect of TNF-alpha was additive to that of epinephrine but was unaffected by a maximal concentration of cholecystokinin. IL-8 did not alter basal or secretagogue (cholecystokinin, epinephrine)-mediated somatostatin release. The stimulatory effect of TNF-alpha (10 ng/mL) was potentiated by the addition of IL-8 (1 nmol/L), inhibited by octreotide and staurosporine, but unaffected by indomethacin. Pretreatment of D cells with TNF-alpha (10 ng/mL) for 24 hours abolished the subsequent stimulatory effect of this cytokine and secretagogues on somatostatin release. CONCLUSIONS: TNF-alpha was shown to regulate somatostatin release from cultured D cells in a divergent manner.

Canine H(+)-K(+)-ATPase alpha-subunit gene promoter: studies with canine parietal cells in primary culture.

H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) is the principal enzyme responsible for the process of gastric acid secretion. This enzyme is expressed in a cell-type-specific manner in gastric parietal cells. To explore the mechanisms regulating its expression, we transfected differentiated canine parietal cells in primary culture with H(+)-K(+)-ATPase-luciferase reporter genes and assessed transcriptional activities. Deletional analysis of the 5'-flanking region of this gene demonstrated a remarkable increment in transcriptional activity associated with a segment between bases -54 to -45 (5' GCTCCGCCTC 3') relative to the transcriptional initiation site. Gel shift assays with competition and supershift analysis demonstrated that this segment is specifically bound by the transcription factor Sp1. A point mutation, eliminating Sp1 binding, diminished basal transcriptional activity by 80%, indicating that this Sp1 binding site is important for constitutive transcriptional activity. Although these studies indicate that Sp1 is required to maintain a high concentration of the H(+)-K(+)-ATPase gene in the parietal cell, its cell-type-specific expression must rely on other elements because Sp1 is a ubiquitously expressed transcription factor.

Canine prosomatostatin: isolation of a cDNA, regulation of gene expression, and characterization of post-translational processing intermediates.

Somatostatin is a tetradecapeptide (SS-14) initially isolated from the hypothalamus that is also found in D cells of the stomach and pancreas where it exerts an inhibitory action on a variety of gastrointestinal functions. Since many of concepts important to an understanding of gastrointestinal physiology are derived from experiments in the dog we examined somatostatin gene expression and post-translational processing in the canine fundus, antrum and pancreas. The canine somatostatin cDNA which is highly homologous to other known mammalian somatostatins was used to examine somatostatin expression in isolated canine fundic D-cells. Somatostatin expression induced by cholecystokinin (10(-8) M) was inhibited by the somatostatin analog, octreotide (10(-7) M). To examine somatostatin processing in the canine gut we noted that synthesis of SS-14 and somatostatin octacosapeptide (SS-28) involves endoproteolytic cleavage of prosomatostatin (proSS) at both paired and single basic amino-acid residues, respectively. Antisera capable of recognizing the amino-terminal residues of SS-28, SS-28(1-14) and SS-28(1-12) were characterized and identified concentrations of SS-28(1-12) but not SS-28(1-14) in the fundus, antrum and pancreas equivalent to those of SS-14. Since previous biosynthetic studies in canine fundic D-cells showed that SS-14 was synthesized without the appearance of a SS-28 intermediate, we hypothesize that proSS is sequentially cleaved at a dibasic site to produce SS-14 followed by monobasic cleavage that results in the formation of SS-28(1-12). Furthermore, equivalent amounts of SS-14 and SS-28(1-12) were co-released from canine fundic D-cells by CCK (10(-8) M) suggesting that the generation of these products occurs within the same regulated pathway of secretion.

Histamine H2 receptor activates adenylate cyclase and PLC via separate GTP-dependent pathways.

Previously, we demonstrated that a single histamine H2 receptor can couple to both the adenosine 3',5'-cyclic monophosphate and inositol 1,4,5-trisphosphate/intracellular Ca2+ signaling pathways in a stimulatory manner. We undertook the present studies to fur her characterize the postreceptor events involved in H2 receptor dual signaling. Histamine H2 receptor-mediated signal transduction was examined in isolated cell membranes prepared from purified canine parietal cells and HEPA cells (rat hepatoma cell line) stably transfected to express the canine H2 histamine receptor cDNA. Histamine dose-dependently stimulated both adenylate cyclase [AC; mean effective concentration (EC50) = 2 x 10(-7) M] and phospholipase C (PLC; EC50 = 3.1 +/- 0.5 x 10(-7) M) activity in an H2-specific and GTP-dependent manner. Cholera toxin pretreatment abolished the stimulatory effect of histamine on PLC activity in isolated membranes without altering binding of the H2 receptor antagonist tiotidine. Anti-Gs alpha dose-dependently inhibited histamine-stimulated AC activity while leaving the effect of this secretagogue on PLC activity unaltered. Although anti-Gq alpha inhibited vasopressin-stimulated PLC activity in HEPA cells and carbachol-stimulated PLC in parietal cells, this antibody did not alter the action of histamine on PLC in the same membrane preparations. Antibody against the NH2 and COOH terminals of the common beta-subunit of heterotrimeric G proteins did not inhibit histamine-stimulated PLC activity. Our studies demonstrate for the the first time that activation of the H2 receptor leads to stimulation of both AC and PLC via separate GTP-dependent mechanisms.

Tumour necrosis factor alpha stimulates gastrin release from canine and human antral G cells: possible mechanism of the Helicobacter pylori-gastrin link.

There is evidence that gastric Helicobacter pylori (Hp) infection promotes duodenal ulceration by releasing gastrin. We therefore asked how Hp releases gastrin. Tumour necrosis factor alpha (TNF-alpha) is up-regulated in Hp gastritis and stimulates hormone release from pituitary cells, so we tested its effect on primary cultures of canine antral G cells and human antral fragments. TNF-alpha pretreatment (100 ng mL-1) of canine G cells significantly increased both basal (by 89%: P < 0.01) and bombesin-stimulated (by 39% P < 0.05) gastrin release. A similar pattern of increase was seen following TNF-alpha (20 ng mL-1) pretreatment of human antral fragments: basal gastrin release was increased by 38% (P < 0.05) and bombesin-stimulated by 26% (P < 0.05). This effect persisted during immunoblockade with anti-somatostatin antibody S6. We propose that TNF-alpha provides the link between Hp infection and gastrin release and thus contributes to duodenal ulceration.

Linkage of [Ca2+]i in single isolated D cells to somatostatin secretion induced by cholecystokinin.

The Ca2+/inositol phospholipid signaling cascade has been implicated in the mechanism by which cholecystokinin (CCK) stimulates gastric somatostatin release, but a direct linkage between intracellular events in gastric D cells and somatostatin secretion has not been established. To address this problem we developed a method for correlating somatostatin release with the measurement of intracellular Ca2+ concentration ([Ca2+]i) in isolated D cells. Resting [Ca2+]i in single D cells was 100 +/- 5.7 nM (means +/- SE, n = 41), and CCK induced a rise in [Ca2+]i in a dose-dependent fashion, producing a maximal stimulatory effect (243 +/- 15% of control, n = 12) at a peptide concentration of 2 x 10(-8) M. The CCK-mediated increase in [Ca2+]i was biphasic, with a rapid, initial transient elevation followed by a sustained plateau. The rise in [Ca2+]i was accompanied by a concomitant increase in release of somatostatin-like immunoreactivity (SLI). Removal of extracellular Ca2+ had no effect on the initial transient elevation in [Ca2+]i induced by CCK but abolished both the sustained plateau in [Ca2+]i and the release of SLI. The selective CCK antagonist L-364, 718 (10(-7) M) inhibited the effects of CCK on both [Ca2+]i and SLI release. The nonspecific Ca2+ channel blocker NiCl2 (10(-3) M) and the L-type Ca2+ channel blocker nifedipine inhibited the sustained rise in [Ca2+]i and the release of SLI but left the initial transient increase in [Ca2+]i unaltered. These results indicate that CCK-stimulated release of SLI from D cells in the gastric fundus is linked to influx of extracellular Ca2+ via L-type Ca2+ channels.

Epithelial response of the rat gastric mucosa to chronic superficial injury.

UNLABELLED: Chronic injury to the healthy gastric mucosa with noxious agents such as aspirin or alcohol induces a progressive strengthening of the stomach wall against these insults. The present study examined the histologic response of the rat gastric mucosa to chronic destruction of the superficial mucosa for one month with hypertonic saline. The number, position and morphology of proliferating, parietal, G and D cells were followed during mucosal injury and one month of recovery. The results showed that chronic injury reduced parietal cell numbers by about 30 percent, particularly in the middle of the mucosal thickness where a clear zone was formed by hypertrophy of mucous neck-like cells. G cells were also reduced by about 50 percent, but there were no changes in D cells. Chronic injury induced a marked increase in the number of antral (+112 percent) and fundic (+250 percent) proliferating cells. CONCLUSION: The rat gastric mucosa responds to chronic superficial injury by down-regulation of acid secretory cells and gastrin secreting cells and an up-regulation of proliferating cells. The appearance of a prominent layer of mucous neck-like cells may indicate a new secretory function for these cells.

Epidermal growth factor inhibits carbachol-stimulated canine parietal cell function via protein kinase C.

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits secretagogue-stimulated gastric acid secretion via an EGF receptor located on parietal cells. The aim of this study was to examine whether this growth factor inhibited carbachol-stimulated acid secretion through a protein kinase C-dependent mechanism. METHODS: The effect of EGF on carbachol-stimulated aminopyrine uptake, inositol trisphosphate formation, and intracellular Ca2+ ([Ca2+]i) in purified cultured parietal cells was studied. The ability of protein kinase A and C inhibitors to alter the inhibitory action of EGF was assessed. EGF-mediated translocation and activation of protein kinase C in parietal cells were determined. RESULTS: EGF dose dependently inhibited carbachol-stimulated aminopyrine uptake in a pertussis toxin-insensitive, genistein (tyrosine kinase inhibitor)--sensitive manner, with a maximal inhibitory effect (37.5% +/- 6.8%) achieved at 10(-7) mol/L. EGF did not significantly inhibit carbachol-stimulated inositol trisphosphate formation and did not alter the initial transient increase or sustained plateau in [Ca2+]i stimulated by this secretagogue. The protein kinase C inhibitors H-7 and staurosporine dose dependently reversed the inhibitory action of EGF, whereas H-89 (protein kinase A inhibitor) failed to alter the effect of EGF. EGF pretreatment increased the translocation of alpha and beta 1 isoforms of protein kinase C and stimulated kinase activity in parietal cells. EGF did not down-regulate the parietal cell muscarinic receptor. CONCLUSIONS: The inhibitory action of EGF on carbachol-stimulated parietal cell activity seems to involve protein kinase C.

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.

Construction of a novel bifunctional biogenic amine receptor by two point mutations of the H2-histamine receptor.

BACKGROUND: H2-histamine receptors mediate a wide range of physiological functions extending from stimulation of gastric acid secretion to induction of human promyelocyte differentiation. We have previously cloned the H2-histamine receptor gene and noted that only three amino acids on the receptor were sufficient to define its specificity and selectivity. Despite only modest overall amino acid homology (34% amino acid identity and 57.5% similarity) between the H2-histamine receptor and the receptor for another monoamine, the beta 2-adrenergic receptor, there is remarkable similarity at their critical ligand binding sites. We hypothesized that, if the specificity and selectivity of both receptors are invested in just three amino acids, it should be possible to convert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites. MATERIAL AND METHODS: We explored the effect of two single mutations in the fifth transmembrane domain of the H2-histamine receptor, which encompasses the sites that determine H2 selectivity. The canine H2 receptor gene was mutated at Asp186 and Gly187 (Asp186 to Ala186 and Gly187 to Ser187) by oligonuceotide directed mutagenesis. The coding region of both the wild-type and mutated H2 receptors was subcloned into the eukaryotic expression vector, CMVneo, and stably transfected into Hepa cells and L cells. The biological activity of histamine and epinephrine on the expressed receptor was examined by measurement of cellular cAMP production and inositol trisphosphate formation. RESULTS: Hepa cells transfected with the Ala186-Ser187 mutant H2 receptor demonstrated a biphasic rise in cAMP in response to epinephrine with an early phase (ED50 approximately 10(-11) M) that could be inhibited by both propranolol and cimetidine. Epinephrine also induced IP3 generation in the same cells, a biological response that is characteristic of activation of the wild-type H2 but not of the beta-adrenergic receptor. L cells transfected with the Ala186-Ser187 mutant H2 receptor also responded to epinephrine in a cimetidine and propranolol inhibitable manner. CONCLUSIONS: We converted the H2-histamine receptor into a bifunctional one that has characteristics of both histamine and adrenergic receptors by two simple mutations. These results support the hypothesis that ligand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. Our studies have important implications on the design of pharmacological agents targeted for action at physiological receptors.

Muscarinic M3 receptor-mediated release of gastrin from canine antral G cells in primary culture.

The functional role of the cholinergic nervous system in regulating gastrin release was investigated using enriched canine antral G cells. Gastrin content was 30.1 +/- 2.9 pmol per well and basal gastrin release was 900 +/- 27 fmol per well (n = 45). Carbachol (10(-8) to 10(-5) M) dose-dependently stimulated gastrin release with a maximal stimulatory response achieved at a concentration of 10(-5) M (330% over basal). To characterize the muscarinic receptor which mediates gastrin release from antral G cells, we examined the effect of three muscarinic receptor antagonists on carbachol-stimulated gastrin release; atropine (nonselective muscarinic receptor antagonist), pirenzepine (M1 muscarinic receptor antagonist) and 4-DAMP (M3 muscarinic receptor antagonist). Atropine (10(-9) to 10(-6) M), pirenzepine (10(-8) to 10(-5) M) and 4-DAMP (10(-9) to 10(-6) M) had no effect on the basal gastrin release. However, carbachol (10(-5) M)-stimulated gastrin release was effectively inhibited by atropine and 4-DAMP with Ki values of 0.48 and 0.66 nM, respectively. Pirenzepine at a high concentration (10(-5) M) also inhibited carbachol-stimulated gastrin release with a Ki value of 46.3 nM. These results suggest that the cholinergic nervous system directly stimulates gastrin release via M3 muscarinic receptors located on antral G cells.

Somatostatin in preterm infants: postnatal changes and response to stress.

To determine the chronology of postnatal somatostatin (SRIF) changes in preterm infants and the relationship of SRIF levels to respiratory and gastrointestinal complications, we evaluated sequential SRIF levels in 62 preterm infants in the first month of life. Weekly preprandial plasma samples were obtained and analyzed for SRIF using a radioimmunoassay. Additional blood samples were obtained at the time of abdominal events. Somatostatin levels were highest in week 2 and gradually declined in weeks 3 and 4 (mean +/- SD pmol/l, SRIF = 92.3 +/- 30.3 in week 2 vs. 79.8 +/- 33.9 in week 3 and 69.7 +/- 54.4 in week 4, p < 0.03). Birth weight, gestational age and sex were not related to initial SRIF levels. Infants with respiratory distress requiring assisted ventilation had significantly higher week 1 SRIF levels compared to infants without respiratory problems (97.9 +/- 22.7 vs. 74.9 +/- 21 pmol/l, p < 0.02). Twenty-one of the 62 infants had gastrointestinal complications. Somatostatin levels preceding (89.0 +/- 25.9 pmol/ 1), during (91.0 +/- 13.3) and after (79.3 +/- 28.6) the gastrointestinal events were not significantly different, nor were they different from SRIF concentrations of age-matched preterm infants without gastrointestinal complications. The results suggest that in preterm infants, postnatal SRIF changes follow a definite pattern with peak concentrations in week 2. Respiratory distress is associated with a significant increase in SRIF. However, subsequent gastrointestinal events do not lead to an increase in SRIF. This lack of SRIF response in gastrointestinal stress may play a role in the pathogenesis of gut injury in the premature neonate.

Molecular basis for somatostatin action: inhibition of c-fos expression and AP-1 binding.

The mechanisms by which somatostatin exerts its widespread inhibitory actions have been investigated extensively but understood only partially. Recent studies have shown that somatostatin can inhibit gene transcription directly. In view of the critical importance of early response genes in induction of gene expression, we examined whether the action of somatostatin might be mediated by inhibition of early response genes. The products of some of these genes, such as c-fos and c-jun, are known to form a heterodimeric transcription factor complex (AP-1) that binds specifically to the consensus sequence TGAC(G)TCA. Accordingly, we examined the effects of somatostatin on c-fos gene expression and on the binding of the AP-1 complex to its specific DNA element using isolated gastric parietal cells and the GH3 pituitary cell line. In both parietal and GH3 cells, c-fos-specific mRNA was increased by agents known to act via both adenosine-3',5'-cyclic monophosphate and Ca(2+)-dependent signaling mechanisms, and octreotide significantly inhibited this response. Pertussis toxin pretreatment (200 ng/ml) reversed the inhibitory effect of octreotide. AP-1 binding activity, assessed by gel shift assays using a 32P-labeled AP-1 oligonucleotide probe, was stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate and serum and inhibited by octreotide treatment. Our observations support the notion that the universal inhibitory action of somatostatin may be mediated by inhibition of expression of early response genes via a pertussis toxin-sensitive inhibitory pathway. This effect appears to lead to decreased binding of regulatory nuclear proteins to their specific DNA elements resulting, presumably, in diminished gene expression.

Interaction of dual intracellular signaling pathways activated by the melanocortin-3 receptor.

We undertook these studies to explore the intracellular signaling mechanisms activated by a newly described human brain melanocortin receptor (hMC3R). Hepa cells transfected with the hMC3R gene responded to stimulation with alpha-melanocyte stimulation hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) with dose-dependent increases in cellular content of cyclic 3',5'-adenosine monophosphate (cAMP) reaching a maximum of over 1500% of control cells at the 10(-8) M dose (EC50 = 10(-11) M). In contrast, the production of [3H]inositol phosphates in cells prelabeled with myo-[2-3H]inositol exhibited a biphasic dose-response curve with increases as high as 155% of basal at 10(-11) M alpha-MSH or ACTH, but beyond that a dose-dependent decrease was observed. The inhibitory component of the dose-response curve could be abolished by pretreatment of transfected cells with the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate (Rp-cAMP) or the protein kinase A inhibitor H-89. Increases in intracellular calcium induced in transfected cells by alpha-MSH in doses ranging from 10(-11) to 10(-7) M could not be observed unless the cells were pretreated with H-89. By replacing the third intracytoplasmic loop of the canine H2-histamine receptor with that of hMC3R the biphasic characteristic of agonist-induced production of [3H]inositol phosphates was conferred to the chimeric receptor. These data indicate that the hMC3R is coupled to both cAMP and inositol phospholipid/Ca(2+)-mediated post-receptor signaling systems and that the latter response is regulated by protein kinase A activity.

Roles of adrenoceptors in isolated canine parietal cells.

Functional roles of adrenoceptors in parietal cells were pharmacologically investigated using isolated canine parietal cells. In the crude membranes obtained from preparations highly purified in parietal cells (> 95% of purity), the specific binding of [3H]dihydroalprenolol (DHA) was observed with a Kd value of 2.9 nM and Bmax of 234 fmol/mg protein, while the specific binding of [3H]prazosin and [3H]rauwolscine were not attained. Propranolol concentration-dependently reduced the specific binding of [3H]dihydroalprenolol with a Ki value of 2.6 nM. Isoproterenol concentration-dependently stimulated [14C]aminopyrine accumulation in preparations enriched in parietal cells (about 70% purity) with the maximum at 10 nM. Isoproterenol increased the content of cyclic AMP in preparations enriched in parietal cells (70%) with the maximum at 100 nM. The isoproterenol-induced stimulatory effect of [14C]aminopyrine accumulation in preparations enriched in parietal cells (70%) was completely abolished by 1 microM propranolol but not by 1 microM phentolamine. In the presence of 1 microM propranolol, 100 microM noradrenaline did not affect carbachol- and histamine-induced [14C]aminopyrine accumulation in preparations enriched in parietal cells (70%). The present study suggests that stimulation of beta-adrenoceptors located on canine parietal cells evokes acid production in a cyclic-AMP-dependent manner. Furthermore, a possibility arises that canine parietal cells are not the site of action of alpha-adrenoceptors in mediating inhibition of gastric acid secretion.