Combined ultrastructural and biochemical study of cellular processing of vasoactive intestinal peptide and its receptors in human colonic carcinoma cells in culture.

Desensitization of human carcinoma colonic cells in culture (HT-29) to vasoactive intestinal peptide (VIP) has been reported previously (C. Boissard, J. C. Marie, G. Hejblum, C. Gespach, and G. Rosselin, Cancer Res., 46: 4406-4413, 1986). In the present study, we have determined the ultrastructural localization of VIP and its receptor after exposure of HT-29 cells to VIP monoiodinated on tyrosyl residue 10 together with the molecular forms and the activity of the internalized VIP receptor. Quantitative electron microscope autoradiography showed that after binding at the cell surface, VIP is internalized in heterogeneous endosomes. Cross-linking experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis were performed in different experimental conditions allowing us to selectively obtain cell surface-associated, internalized, or recycled receptors. No detectable alteration of the labeled VIP-receptor complex occurred during the internalization and recycling processes. Furthermore, a loss of the forskolin potentiation of the VIP-induced stimulation of adenylate cyclase was observed after VIP exposure. This feature was time and temperature dependent as was the VIP-induced loss of cell surface receptors, indicating that the internalized VIP receptor is dissociated from the adenylate cyclase.

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.

Stimulatory transducing systems in pancreatic islet cells.

We have determined the cellular distribution of different alpha subtypes of G proteins and adenylyl cyclase (AC) isoforms in endocrine, exocrine, and established pancreatic cell lines. VIP, PACAP, and tGLP-1 receptor proteins are expressed to varying extents in A and B cells, whereas the expression of G alpha subunits is cell specific. Thus, G(olf) alpha is detected in normal rodent B cells and immortalized pancreatic B cell lines, whereas Gs alpha is more ubiquitously expressed. The cellular density of AC isoforms labeling (I, II, III, IV, V/VI) is also islet cell-specific and their distribution is age- and species-dependent. The identification of numerous signaling molecule subtypes, together with the discovery of their specific subcellular distribution, will help the functional characterization of their intraregulatory pathways, leading to the extrusion of insulin or glucagon secretory granules, and those leading to differentiation and apoptosis of islet cells.

VIP inhibits histamine-induced ultrastructural changes related to acid secretion by parietal cells.

We have studied the in vitro effect of VIP and histamine on ultrastructure of the parietal cells in isolated guinea pig fundic glands. The morphological changes induced by histamine in the parietal cells can be compared to those observed after histamine stimulation in vivo or in vitro on gastric mucosa preparations. In contrast, VIP incubation did not produce the ultrastructural changes related to gastric acid secretion, in resting parietal cells. Pretreatment of the glands by VIP resulted in a remarkable suppression of the histamine effect, since the parietal cells assumed an almost resting state. The data (1) indicate that the parietal cells in isolated gastric glands of the guinea pig retain in vitro the capacity to undergo the ultrastructural changes that are related to acid secretion in vivo after histamine or cAMP and (2) suggest that VIP is an inhibitor of histamine-induced gastric acid secretion in the guinea pig. It is proposed that VIP could act directly on the parietal cell via cAMP-phosphodiesterase activation, or indirectly via gastric somatostatin and/or prostaglandin secretions, inhibiting the H2 receptor-cAMP system of the parietal cell.

Ultrastructural characteristics of developing eosinophil leukocytes in human bone marrow during acute leukemia: evidence for extracellular granule release from human eosinophils.

Developing eosinophils from the bone marrow of a patient with acute "eosinophilic" leukemia were characterized by electron microscopy. It was suggested that the first sequential step in granule formation occurred at the level of the endoplasmic reticulum without actual participation of the Golgi complex. Progressive densification of the former profiles, presumably mediated by Golgi vesicles, resulted in the formation of dense immature granules. Ultrastructural observations of the "leukemic" eosinophils which were generally arrested at an intermediate stage of maturation revealed also large vacuoles containing sequestered immature granules, without any indication of phagocytic activity. Morphological evidence that has been accumulated indicates that the membrane of these vacuoles fused with the cell membrane, thus being in contact with the extracellular space. These profiles strongly suggested that granules and/or granule-associated material were secreted by developing bone marrow eosinophils.

Ultrastructural analysis of VIP internalization in rat beta- and acinar cells in situ.

We perfused the pancreas with 125I-labeled vasoactive intestinal peptide (VIP) to follow the concomitant distribution of radioactivity in beta- and acinar cells as a function of time. This distribution was quantitated by computer-assisted analysis of high-resolution video autoradiographs. Density labeling was expressed as normalized specific activity (disintegration density per volume density). Immediately after a 4-min perfusion of 125I-VIP, labeling in beta-cells was mainly concentrated on the cell surface and peripheral tubules and vesicles. After three 30-s pulses of 125I-VIP, separated by intervals of 3.5 min of buffer perfusion, lysosome-like structures were heavily labeled. When VIP internalization was prolonged, labeling was similar to that observed with the 4-min perfusion, indicating a high VIP disposal rate in the lysosome-like structures. In acinar cells, labeling persisted on the surface and the early vacuolar system. We conclude the following: 1) an active endocytotic system, linked to the transport and sorting of a neuromediator, is present in beta-cells; and 2) the differences between the distribution of labeling in acinar and beta-cells suggest that the regulation of VIP internalization is tissue specific.

Hairy cell leukemia: a clinical, immunological and ultrastructural study. New data for the origin of the hairy cell.

Seven patients presenting with the characteristic clinical and laboratory findings of HCL were investigated mainly in order to find the origin of the HC with histochemical, electron microscopic and immunologic techniques. The results we obtained are somewhat contradictory, as in the recent literature, if the HC's are to the considered as being exclusively of lymphocytic or monocytic origin. The data however indicate that HCL is not of one cell type but two. One of these cells has characteristics of a lymphocyte. The other one, an ill defined cell, has mixed characteristics of both the monocyte and lymphocyte series. This hairy cell may represent a new cell line.

Dynamics of pancreatic cell growth and differentiation during diabetes reversion in STZ-treated newborn rats.

Cellular processes underlying ontogenesis and regression of streptozotocin (STZ)-induced diabetes in newborn rats were investigated at the most severe stage of diabetes at day 3 and after recovery of normoglycemia at day 8 by immunocytochemistry and quantitative analysis. A previously unknown endocrine cell type subpopulation (PEPS) was identified. It was characterized by granule polymorphism, coexpression of insulin and glucagon immunoreactivity, and a proliferative capacity transiently higher than in B cells. In STZ-treated rats at day 3, B cell mass decreased 14-fold, whereas PEPS cells were unaffected. The islet mass was restored to 55.7% by day 8, with a concomitant appearance of numerous small islets contiguous to small ducts. B cell mass increased by 6.9-fold compared with 1.8-fold in control rats, although proliferative capacities remained similar. Proliferation dropped considerably by day 8, preventing complete B cell mass recovery in STZ-treated rats. STZ-induced neonatal diabetes thus stimulates neogenesis of islets close to ducts and proliferation of PEPS cells. Those partially differentiated islet cells appear to be on the differentiation pathway of stem cells to fully differentiated B cells.

Effects of macrophage activity on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae: an in vitro cytotoxicity and ultrastructural study.

The effect of the activity of macrophages on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae was studied in vitro. Macrophages present in peritoneal exudates from mice genetically selected for high and low antibody production (HL and LL, respectively) showed an inverse cytotoxic effect. Cells from HL mice were ineffective, whereas cells from LL mice had a very high killing capacity. Ultrastructural studies of cells after incubations of up to 36 h supported these observations. Furthermore, peritoneal macrophages from congenitally athymic (nu/nu) mice showed a higher killing potential than cells from thymus-bearing littermates (+/nu) mice. The activity of the latter cells could be increased by in vitro pretreatment of the mice with Calmette-Guérin bacillus, a well-known macrophage stimulating agent. The results indicate that macrophages, although not the only effector cells, may play an important role in the defence against T. spiralis newborn larvae.

[Demonstration of VIP receptors in B cells of the pancreas by quantitative ultrastructural autoradiography]. / Mise en évidence des récepteurs au VIP dans les cellules B du pancréas par autoradiographie ultrastructurale quantitative.

Using quantitative electron microscopic autoradiography we demonstrated the existence of specific VIP receptors in B cells of the pancreatic islets. The presence of those receptors strongly suggests that the neuromediator can induce insulin release by direct stimulation of B cells.