Angiotensin I converting enzyme in gastric mucosa of the rabbit: localization by autoradiography, immunofluorescence, and immunoelectron microscopy.

To localize angiotensin converting enzyme (ACE) in the fundic mucosa of the rabbit, we used autoradiography with the ACE inhibitor [3H]-trandolaprilate and post-embedding immunocytochemical techniques with a goat anti-rabbit ACE, using fluorescence and electron microscopy. Autoradiographic localization of [3H]-trandolaprilate in rabbit fundus sections shows that ACE is present in the fundic mucosa and mainly in the gland area. Fundic mucosa was fixed with 4% formaldehyde and embedded in Lowicryl K4M. Semi-thin (1 micron) or thin sections (800-900 A) were stained with anti-rabbit ACE followed by fluorescein isothyocyanate-labeled rabbit anti-goat IgG or protein A-gold reagent, respectively. Label was present on endothelium of all blood vessels running through the mucosa. Label was prominently localized in the granules of mucous surface and neck cells and on the granules of chief cells. The intracellular localization of ACE, and particularly its intragranular presence within chief and mucous cells, suggests that the enzyme, at the fundic level, is involved in the intragranular processing of a peptide, the nature of which remains to be determined.

Localization of angiotensin-converting enzyme (ACE) mRNA in rabbit gastric mucosa by in situ hybridization.

In a previous study we showed, by immunohistochemical analysis on rabbit fundic mucosa, that in addition to its usual presence on the luminal plasma membrane of endothelial cells, angiotensin converting-enzyme (ACE) was localized inside granules of surface and neck mucous cells and within granules of chief cells. The aim of the present study was to localize ACE mRNA in cells of the rabbit fundic mucosa by in situ hybridization with a 35S-labeled probe. This probe was a cDNA fragment (406 BP) encoding a portion of the rabbit ACE mRNA obtained by reverse transcription followed by polymerase chain reaction on total RNA extracted from fundic mucosa. ACE mRNAs were detected in mucous and chief cells and in endothelial cells of the mucosal vasculature. These results are in complete agreement with our prior studies which showed by immunohistochemical analysis that ACE is present in these cells. Our findings therefore suggest that ACE previously detected in epithelial cells of the rabbit gastric mucosa is actually synthesized within these cells.

Expression of angiotensin I converting enzyme mRNA in rabbit gastric epithelial cells.

Angiotensin I converting enzyme (ACE) is a dipeptidyl carboxypeptidase synthesized by endothelial cells from many vascular beds as well as by extravascular tissues. Two forms of ACE have been characterized, a pulmonary form and a testicular form. Previously, in the gastrointestinal tract, we localized ACE in the rabbit gastric fundic tissue. In the present study, Northern blot analysis demonstrated the expression of a 5 kb ACE mRNA in fundic mucosa, identical in size to pulmonary ACE mRNA. In order to confirm the epithelial origin of this ACE, we have purified fundic epithelial cells by a flow cytometry technique by which endothelial cells were excluded and the population was enriched in intermediate and chief cells. Using reverse transcription and polymerase chain reaction with specific oligonucleotides, we have amplified from the enriched fundic epithelial cell RNA a 874 bp fragment, the restriction map of which is identical to that of rabbit lung. These findings demonstrate that in gastric mucosa ACE is expressed in fundic epithelial cells and seems to be similar to the pulmonary form.

Evidence for autocrine growth stimulation by a gastrin/CCK-like peptide of the gastric cancer HGT-1 cell line.

Gastrin has been shown to promote the growth of some colonic tumor cell lines. To evaluate the involvement of this hormone in the proliferation of gastric tumors, we studied the effects of gastrin/CCK-receptor antagonists (L365,260 and L364,718), proglumide and C terminal-specific gastrin antibodies on the human gastric adenocarcinoma cell line HGT-1. L365,260, but not L364,718, dose-dependently inhibited cell proliferation (72% after 4 days at 10 nM) and [3H]thymidine incorporation (68% after 2 days at 10 nM) in serum-free medium. No cytotoxic effects of proglumide or L365,260 on this cell line were detected. Proglumide inhibited cell proliferation in serum-free medium (40% and 66.5% after 2 and 4 days of treatment; IC50 = 1.4 mM) and in 5% fetal calf serum (FCS)-supplemented medium (30% and 22% after 2 and 4 days of treatment; IC50 = 3.25 mM). [3H]Thymidine incorporation was also inhibited by proglumide in serum-free medium (IC50 = 2.3 mM) and 5% FCS-supplemented medium (IC50 = 3.35 mM). Gastrin did not induce cell proliferation or increase [3H]thymidine incorporation and no high-affinity gastrin binding sites were observed. However, C terminal-specific gastrin antibodies, even at low concentration, caused a dramatic decrease in both cell number (IC50 = 1:4000 antiserum dilution) and [3H]thymidine incorporation (IC50 = 1:400 antiserum dilution) in the HGT-1 cell line. In addition, immunofluorescence analysis revealed that these antibodies specifically bind HGT-1 cells and radioimmunoassay analysis confirms the presence of gastrin/CCK-like peptide in cell extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of angiotensin I-converting enzyme in the human gastric HGT-1 cell line.

We have previously shown that angiotensin I-converting enzyme (ACE) was expressed by epithelial cells of the rabbit gastric mucosa. In a search to obtain a cell model to study the regulation of ACE expression of gastric origin and its relationship with gastrin-cholecystokinin peptides, which have been proposed as ACE substrates, we investigated whether the HGT-1 human gastric cell line, which expresses gastrin, could also express ACE, using enzymatic and immunodetection methods as well as Northern-blot analysis and polymerase chain reaction. Results show that HGT-1 cells expressed a protein with a molecular weight of 130-140 kDa whose enzymatic and immunological properties were identical to those of ACE. More than 80% of ACE activity was found to be ectoenzymatic. However, immunocytochemical localization has mainly shown an intracellular localization, suggesting that most of intracytoplasmic ACE was not enzymatically active. In addition, Northern-blot analysis and polymerase chain reaction showed that the mRNA encoding that protein displayed a size and a sequence identical to those of somatic ACE. It therefore appears that the HGT-1 cell line could be a useful model to study both the regulation of gastric ACE and its interactions with gastrin-cholecystokinin peptides.

Endothelin expression in human megakaryoblastic leukemia cell lines and normal platelet precursors.

The aim of the present investigation was to determine whether endothelin (ET) could be expressed in and released from the human leukemia megakaryoblastic cell lines HEL, MEG-01, DAMI and the normal human platelet progenitors. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) on total RNA isolated from the cells, we amplified a cDNA of the expected size (453 bp). Southern-blotting hybridization revealed that RT-PCR products from the cell lines were specific of ET-1 mRNA. Immunocytochemical analyses highlighted immunoreactive ET-1 in the cytoplasm of these cells which also released the mature peptide. ET-1 release from the three cell lines was increased by thrombin exposure. Although MEG-01 cells express ET receptors, ET-1, the selective ETB agonist sarafotoxin 6C and the non-selective ET-receptor antagonist PD 142893 showed no proliferative or antiproliferative action in basal or stimulating medium. This indicated a lack of autocrine ET-mediated effect on growth. These results demonstrate for the first time that human megakaryoblastic leukemia cell lines and normal bone marrow platelet precursors express ET-1 mRNA and release the mature peptide.

Neurogenic modifications induced by substance P in an organ culture of human skin.

Neurogenic inflammation of the skin observed after topical application of an irritant substance or environmental stimulation induces vascular changes and the production of inflammatory mediators. Substance P (SP) is one of the main neuropeptides which trigger an inflammatory response in the skin. So, with the aim to develop an alternative method to study neurogenic inflammation of the skin, we used an organ culture of human skin. SP was added onto epidermis or directly to culture medium in an attempt to reproduce ex vivo the effects described in vivo. Even disconnected from systemic blood circulation, in skin fragments in culture, we observed dose-dependent edema, vasodilation and extravasation of lymphocytes and mast cells through the microvascular wall. Moreover, the release of proinflammatory mediators interleukin 1alpha and tumor necrosis factor alpha was evidenced.

Abnormal deposition of extracellular matrix proteins by cultured smooth muscle cells from human varicose veins.

The aim of the present study was to verify whether the modifications of the extracellular matrix, described in varicose veins, are also present in cultures of smooth muscle cells from human varicose veins. The accumulation of collagen type III and fibronectin was determined by immunofluorescence in cultures of smooth muscle cells at passage 2-3 during the proliferation phase. After 5 days of culture, the immunostaining of both collagen type III and fibronectin was weaker in cells from varicose than in those of control veins while the expression of collagen type III and fibronectin messenger ribonucleic acids was not significantly different. Collagen type I and III synthesis were quantified by tritiated proline incorporation in control and varicose cell layers at postconfluence. Collagen type I deposition was similar in both types of cell layers while collagen type III was decreased in cell layers from varicose veins. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) were also quantified by enzyme immunoassays in supernatants from smooth muscle cell cultures at postconfluence. No significant difference was observed in the synthesis of any of the MMPs (-1, -2 and -9) or their inhibitors (-1 and -2) tested. These data illustrate that smooth muscle cells cultured from varicose veins deposit less collagen type III and fibronectin than control cells despite comparable levels of mRNAs for these proteins suggesting dysregulation of posttranslational steps in the synthesis of both proteins by smooth muscle cells from varicose veins.