Kerstin Dehne

Exendin-4 and exendin-(9–39)NH2: agonist and antagonist, respectively, at the rat parietal cell receptor for glucagon-like peptide-1-(7–36)NH2

Exendin-4 is a novel peptide from Heloderma suspectum venom which is 53% homologous with glucagon-like peptide-1 GLP-1-(7-36)NH2, a stimulant of cAMP-dependent H+ production in rat parietal cells. It was the aim of the present study to determine whether this effect of GLP-1-(7-36)NH2 is shared by exendin-4, and whether the responses to either peptide are blocked by exendin-(9-39)NH2, a competitive specific exendin receptor antagonist. In enriched rat parietal cells H+ production was measured indirectly by [14C]aminopyrine accumulation. cAMP production was determined by radioimmunoassay. [125I]GLP-1-(7-36)NH2 was prepared using chloramine T followed by high pressure liquid chromatography (HPLC) purification. Exendin-4 (10(-12) - 10(-8) M) stimulated [14C]aminopyrine accumulation in a concentration-dependent manner (EC50 = 7.6 x 10(-11) M). At the maximally effective concentration (10(-9) M) exendin-4 was as effective as GLP-1-(7-36)NH2 reaching 70-80% of the response to 10(-4) M histamine. Likewise, exendin-4 (10(-11) - 10(-7) M) stimulated parietal cell cAMP production up to 2.8-fold. Maximal stimulation by exendin-4 of [14C]aminopyrine accumulation was not affected by ranitidine (10(-4) M), but was concentration-dependently reduced by exendin-(9-39)NH2 (10(-11) - 10(-7) M). At the maximal concentration, exendin-(9-39)NH2 completely abolished the responses to 10(-9) M exendin-4 and to 10(-9) M GLP-1-(7-36)NH2 while not altering stimulation by 10(-4) M histamine. Binding of [125I]GLP-1-(7-36)NH2 to enriched parietal cells was displaced by exendin-4 (Ki = 4.6 x 10(-10) M) as well as by exendin-(9-39)NH2 (Ki = 4.0 x 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

beta(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter

ABSTRACT Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that β3-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition β3-integrin regulates u-PAR expression. Overexpression of β3-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (−1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced β3-integrin expression. A region between −398 and −197 bp of the u-PAR promoter was critical for β3-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at −248 bp substantially impaired the ability of β3-integrin to downregulate the u-PAR promoter, suggesting that thePEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when β3-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the β3A-integrin isoform but not by other β3-integrin isoforms and required the cytoplasmic membrane NITY759 motif. Moreover, overexpression of the short but not the long isoform of the β3-integrin adapter protein β3-endonexin blocked u-PAR promoter activity through the PEA3/etsbinding site. Thus, besides the physical interaction of β3-integrin and u-PAR at the cell surface, β3 signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

Identification and functional importance of IL-1 receptors on rat parietal cells

We studied the expression of interleukin-1 (IL-1) receptors and the effect of IL-1beta on the function of highly enriched (>97%) rat parietal cells. RT-PCR of parietal cell poly(A)+ RNA with primers specific for the rat IL-1 receptor revealed a single 547-kb PCR product highly homologous to the published sequence of the IL-1 receptor. Northern blot analysis of poly(A)+ RNA of rat parietal cells and brain revealed a single RNA species of 5.7 kb. Cytochemistry of parietal cell IL-1 receptor was performed with biotinylated recombinant human IL-1beta, visualized by avidin-coupled fluorescein. Corresponding to the high degree of parietal cell enrichment, 95% of the cells stained positive. Basal H+ production ([14C]aminopyrine accumulation) was not changed by IL-1beta (0.25-100 pg/ml) nor was the response to histamine or carbachol when added simultaneously with the cytokine. However, when parietal cells were preincubated with IL-1beta (0.5-5 pg/ml) for 10 min before the addition of histamine or carbachol, the response to these secretagogues was reduced by 35 and 67%, respectively. Inhibition by IL-1beta was fully reversed by the human recombinant IL-1 receptor antagonist. Preincubation of parietal cells with IL-1beta failed to alter histamine-stimulated cAMP production but markedly inhibited carbachol-induced formation of D-myo-inositol 1,4, 5-trisphosphate. In fura 2-loaded, purified parietal cells, 10 min preincubation with IL-1beta dramatically reduced the initial transient peak elevation of intracellular Ca2+ concentration in response to carbachol. We conclude that rat parietal cells express IL-1 receptors mediating inhibition of H+ production. The antisecretory effect of IL-1beta may contribute to hypoacidity secondary to acute Helicobacter pylori infection or during chronic colonization by H. pylori preferring the fundic mucosa.

Oxyntomodulin: A cAMP-Dependent Stimulus of Rat Parietal Cell Function via the Receptor for Glucagon-Like Peptide-1 (7-36)NH2

We have previously shown that in highly enriched rat gastric parietal cells the intestinal peptide hormones oxyntomodulin and glucagon-like peptide-2 (GLP-2) compete for receptor-binding with glucagon-like peptide-1 (GLP-1), a potent cAMP-dependent stimulus of H+ production in vitro. It is, however, unknown whether oxyntomodulin and GLP-2 elicit a biological response by interacting with the GLP-1 receptor. Therefore, we used enriched rat parietal cells to investigate the effects of both hormones on the production of cAMP and H+ ([14C]aminopyrine accumulation). Both parameters were stimulated by oxyntomodulin in a concentration-dependent manner. EC50 values were 6.2.10(-8) and 2.5.10(-7) M oxyntomodulin for stimulation of H+ and cAMP production, respectively. The maximally effective concentrations for stimulation of [14C]aminopyrine accumulation and cAMP production were 1.10(-6) and 1.10(-5) M oxyntomodulin, respectively. At these concentrations oxyntomodulin was nearly as effective as 10(-4) M histamine and equally effective as 10(-8) M GLP-1 (7-36)NH2. In the enriched parietal cell preparation there was no immunocytochemical evidence of contaminating D cells. Accordingly, the responses to oxyntomodulin and GLP-1 (7-36)NH2 were not augmented by incubating the cells in the presence of a polyclonal anti-somatostatin antibody. [14C]Aminopyrine accumulation in response to oxyntomodulin was inhibited by the GLP-1 (7-36)NH2 receptor antagonist, exendin (9-39)NH2, but not by the H2-receptor antagonist, ranitidine. Oxyntomodulin and carbachol acted additively to stimulate [14C]aminopyrine accumulation. GLP-2 (10(-7) to 10(-5)M) was without effect on basal H+ and cAMP production; however, at 10(-5) M GLP-2 markedly inhibited oxyntomodulin-stimulated [14C]aminopyrine accumulation. It is concluded that, by interacting with parietal cell receptors for GLP-1 (7-36)NH2, oxyntomodulin, but not GLP-2, directly stimulates H+ production by activating the adenylate cyclase.

Downregulation of a mitogen-activated protein kinase signaling pathway in the placentas of women with preeclampsia

Objective To investigate whether the activity of the three mitogen-activated protein kinases (Jun aminoterminal kinase, extracellular regulated kinase, and p38) is altered in placental tissue of women with preeclampsia and hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. Methods Placental activity (measured by immunoprecipitation-kinase assay) and protein expression (measured by western blot) of Jun aminoterminal kinase, extracellular regulated kinase, and p38 mitogen-activated protein kinase were measured in four groups of eight women each with preeclampsia, HELLP syndrome, and normal vaginal or cesarean deliveries. To further characterize the Jun aminoterminal kinase signal transduction pathway, phosphorylation of c-Jun, a downstream effector of Jun aminoterminal kinase–mitogen-activated protein kinase, was analyzed by western blotting, and the activity of Rac1, an upstream activator of the Jun aminoterminal kinase signaling pathway, was determined by pull-down assay. Results The activity of Jun aminoterminal kinase was significantly lower in placentas of women with preeclampsia or HELLP syndrome compared with those who had normal vaginal or cesarean delivery, whereas levels of Jun aminoterminal kinase protein expression were similar. Phosphorylation of the transcription factor c-Jun and Rac1 activity also were significantly lower in women with preeclampsia and HELLP than in controls. p38 mitogen-activated protein kinase activity was significantly higher in women with preeclampsia than with HELLP syndrome. There was no change in extracellular regulated kinase activity or protein expression between subgroups. Conclusion In placentas of women with preeclampsia or HELLP syndrome, a Rac1-Jun aminoterminal kinase-c-Jun-dependent signal transduction pathway was downregulated.

Pertussis toxin-sensitive inhibition of glucagon-like peptide 1-stimulated acid production by epidermal growth factor and transforming growth factor α in rat parietal cells

We have recently shown that the intestinal hormone glucagon-like peptide-1 (GLP-1)-(7-36) amide is a cAMP-dependent stimulant of rat parietal cell H+ production. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) are known to inhibit histamine-stimulated parietal cell function by reducing cAMP production in a pertussis toxin-sensitive manner. Pertussis toxin blocks Gi alpha, the inhibitory subunit of adenylate cyclase, thereby preventing inhibitors from acting via Gi alpha. Therefore, we used pertussis toxin as a tool to determine whether EGF and TGF alpha inhibit GLP-1-stimulated parietal cell function via Gi alpha. In enriched (76 +/- 4%) rat parietal cells [14C]aminopyrine accumulation and cAMP production were maximally stimulated by GLP-1-(7-36) amide (10(-8) and 10(-7) M, respectively) or by histamine (10(-4) and 10(-3) M, respectively). EGF and TGF alpha (10(-13)-10(-7) M) caused concentration-dependent inhibition of GLP-1-stimulated parietal cell function. Maximal inhibition (33% and 37% of the response to GLP-1-(7-36) amide was observed at 10(-8) M EGF and 10(-9) M TGF alpha, respectively. There was a close correlation (r = 0.83; P < 0.05; n = 7) between the inhibition by EGF and TGF alpha of [14C]aminopyrine accumulation and the fall in cAMP production in GLP-1-stimulated parietal cells. The identical concentrations of both growth factors which maximally reduced GLP-1-stimulated parietal cell function inhibited [14C]aminopyrine accumulation in response to histamine by approximately 30%.(ABSTRACT TRUNCATED AT 250 WORDS)