Growth factor effects on apoptosis of rat gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric epithelium that show increased density during chronic atrophic gastritis. The current study determined cell number and apoptosis of isolated rat ECL cells in response to several growth factors. Isolated ECL cells from fundic mucosa (enrichment >90%) were grown in serum-free medium over 2-5 days. Cell number was determined by mitochondrial formazan production; apoptosis was measured by Tdt-mediated dUTP nick end labeling reaction and DNA fragmentation-based enzyme-linked immunosorbent assay. Immunocytochemistry and RT-PCR demonstrated the presence of epidermal growth factor receptor, neuronal growth factor receptor (type 1), and fibroblast growth factor (FGF) receptor (type 1). Gastrin (EC50, approximately 2 pM), transforming growth factor-alpha (TGF alpha; 10-30 ng/ml), and basic FGF (bFGF; 1-10 ng/ml) increased the total number of cultured ECL cells. bFGF augmented the gastrin (1 pM)-induced response. Beta-neuronal growth factor (10 ng/ml) and bFGF (2 ng/ml) decreased the programed death of ECL cells. Interleukin-1beta (100 pg/ml, 24 h) stimulated apoptosis 2- to 3-fold in ECL cells, and simultaneous incubation with TGF alpha (20 ng/ml) or bFGF (2 ng/ml) significantly inhibited this effect. ECL cells express specific receptors for gastrin, epidermal growth factor, neuronal growth factor, and FGF. bFGF prolonged ECL cell survival by inhibiting spontaneous apoptosis. Our data further indicate that TGF alpha and bFGF increase ECL cell number by inhibiting cytokine-induced programed cell death.

The genetics of osteoarthritis in STR/ort mice.

OBJECTIVE: The complex genetics of osteoarthritis (OA) are still poorly defined. To circumvent the problems of genetic and environmental diversity hampering the analysis in humans, we investigated quantitative trait loci (QTL) associated with murine OA in the STR/ort strain which spontaneously develops osteoarthritic changes of the knee joints, overweight and elevated serum cartilage oligomeric matrix protein (COMP) levels. METHODS: Two hundred and seventy six male F2 intercross (STR/ortxC57BL/6) animals were genotyped using 96 microsatellite markers and phenotyped by analyzing weight, serum COMP levels and osteoarthritic changes of the knee joints. Quantitative trait analyses were performed using the R/qtl software. RESULTS: Elevated weight, serum COMP levels and osteoarthritic changes of the knee joints in the F2 generation compared to C57BL/6 parental animals confirm Mendelian inheritance. Quantitative trait analyses revealed three weight-, one serum COMP- and one OA-locus. CONCLUSIONS: The weight-QTL coincide with previously described genes and QTL involved in fatty acid metabolism and offer a plausible explanation for the observed phenotype in STR/ort mice. The exact match of the COMP-QTL and the COMP gene itself suggests a regulatory polymorphism to account for elevated serum levels in STR/ort mice and questions the robustness of serum COMP as a prognostic marker in human knee OA. The newly identified QTL associated with degenerative changes of the knee joints support the concept of OA resulting from a defective chondrocyte metabolism and/or altered apoptosis rate. However, we also discuss the unlikelihood of one QTL being responsible for OA in STR/ort mice and the inherent limitations of microsatellite analyses for complex genetic diseases.

Sexual dimorphism in the osteoarthritis of STR/ort mice may be linked to articular cytokines.

BACKGROUND: STR/ort mice spontaneously develop degenerative changes of the knee joints resembling human osteoarthritis (OA), with the males being more severely affected than the females. OBJECTIVE: To analyse the early changes leading to OA by examining the articular cytokine expression and degenerative changes in STR/ort mice. METHODS: 122 STR/ort mice of both sexes aged between 2 and 15.5 months were included. Thin sections of the knees were analysed for osteoarthritic changes by haematoxylin/eosin staining. The articular cytokine expression was investigated by immunohistochemical staining using monoclonal antibodies specific for interleukin (IL)6, tumour necrosis factor alpha, transforming growth factor beta1 (TGFbeta1), IL1beta, IL4, and IL10, respectively. RESULTS: Both cartilage degeneration and articular cytokine expression differ between the sexes. The protection from cartilage degeneration in the female mice correlates with an increased expression of TGFbeta1 and IL4 at 2 months of age. CONCLUSION: The increased expression of TGFbeta1 and IL4 in young STR/ort female mice suggests that the sexual dimorphism is mediated through the articular expression of cytokines involved in cartilage metabolism.

Functional impairment of rat enterochromaffin-like cells by interleukin 1 beta.

BACKGROUND & AIMS: Histamine-producing enterochromaffin-like (ECL) cells play an integrative role in the regulation of acid secretion. Decreased mucosal histamine concentrations and increased levels of interleukin (IL) 1 beta, IL-6, and IL-8 have been detected in the gastric mucosa inflamed with Helicobacter pylori. The aim of this study was to investigate the response of isolated ECL cells to these cytokines. METHODS: Enriched rat gastric ECL cells (85%-95%) were cultured for 2-4 days. RESULTS: Polymerase chain reaction showed IL-1 and IL-6, but not IL-8 receptors, in ECL cell complementary DNA. Positive receptor staining with biotinylated IL-1 beta corresponded to ECL cell enrichment (92%). IL-6 and IL-8 had no effect on histamine secretion. IL-1 beta (2 U/mL) stimulated basal histamine secretion and nitric oxide production within 60 minutes and cyclic guanosine monophosphate production within 20 minutes. Pretreatment for 20 minutes with IL-1 beta (2 U/mL) attenuated gastrin-stimulated histamine secretion by 40%-50%, reversed by the IL-1 receptor antagonist (10 U/ mL). Pretreatment for 20 minutes with IL-1 beta (2 U/mL) completely inhibited gastrin-stimulated (1 nmol/L) histidine decarboxylase activity. IL-1 beta (2 U/mL, 60 minutes) increased lactate dehydrogenase release to 25% of cell content. Cells pretreated with IL-1 beta did not respond to gastrin after a further 48-hour culture and showed decreased histamine content. CONCLUSIONS: ECL cells appear to express IL-1 receptors. IL-1 beta causes sustained functional impairment of ECL cells in vitro.

IL-1beta-induced apoptosis in rat gastric enterochromaffin-like cells is mediated by iNOS, NF-kappaB, and Bax protein.

BACKGROUND & AIMS: Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric mucosa. Previous studies have shown that the proinflammatory cytokine interleukin (IL)-1beta present during chronic gastritis inhibits histamine synthesis in ECL cells and leads to sustained functional impairment. This study investigated the effects of IL-1beta on ECL cell apoptosis and the related signal-transduction mechanisms. METHODS: ECL cells were isolated by pronase digestion and a combination of elutriation, gradient centrifugation, and 48-hour culture (purity >/=90%). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling reaction and cell death detection enzyme-linked immunosorbent assay. RESULTS: IL-1beta (100 pg/mL) increased the rate of programmed cell death 2-3 fold in ECL cells after 24 hours of incubation (total of 12%-14%). This effect was completely inhibited by the NF-kappaB inhibitor, proteasome inhibitor I, and the nitric oxide synthase inhibitor (iNOS) N(G)-monomethyl-L-arginine (10(-4) mol/L), but not by the caspase 3 inhibitor, Asp-Glu-Val-Asp-CHO. Western blot analysis, reverse-transcription polymerase chain reaction (PCR), and in situ PCR showed that IL-1beta induced gene expression (after 2-4 hours) and protein synthesis (6-18 hours) of the iNOS isoform in ECL cells. Bax protein expression was increased in response to IL-1beta. In contrast, bcl-2 gene expression was increased in response to basic fibroblast growth factor, which has been shown to counteract IL-1beta- induced apoptosis. CONCLUSIONS: These data suggest that IL-1beta induces programmed cell death in isolated rat ECL cells via activation of NF-kappaB, iNOS, and the Bax protein.

The mechanism of histamine secretion from gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells play a pivotal role in the peripheral regulation of gastric acid secretion as they respond to the functionally important gastrointestinal hormones gastrin and somatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the key stimulus of histamine release from ECL cells in vivo and in vitro. Voltage-gated K(+) and Ca(2+) channels have been detected on isolated ECL cells. Exocytosis of histamine following gastrin stimulation and Ca(2+) entry across the plasma membrane is catalyzed by synaptobrevin and synaptosomal-associated protein of 25 kDa, both characterized as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein. Histamine release occurs from different cellular pools: preexisting vacuolar histamine immediately released by Ca(2+) entry or newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized by cytoplasmic HDC and accumulated in secretory vesicles by proton-histamine countertransport via the vesicular monoamine transporter subtype 2 (VMAT-2). The promoter region of HDC contains Ca(2+)-, cAMP-, and protein kinase C-responsive elements. The gene promoter for VMAT-2, however, lacks TATA boxes but contains regulatory elements for the hormones glucagon and somatostatin. Histamine secretion from ECL cells is thereby under a complex regulation of hormonal signals and can be targeted at several steps during the process of exocytosis.