Noninvasive orthogonal polarization spectral imaging as applied to microvascular studies in mice.

In vivo observations of the mouse microcirculation can hardly be performed due to technical difficulties, limiting the knowledge that could be obtained from gene manipulated mice models. The aim of the present study was to check the applicability of a novel optical system, the orthogonal polarization spectral technology, to study the mouse microcirculation. In anaesthetized mice, the spinotrapezius muscle microcirculation was observed in situ. The diameter of precapillary arterioles was measured before and after a pharmacological or hormonal stimulation. High-contrast images of the muscle microcirculation were obtained and significant vasodilatation of arterioles was observed after topical applications of acetylcholine, sodium nitroprusside, and insulin. As compared to conventional techniques, orthogonal polarization spectral imaging makes it possible to assess and study microvascular beds in mice, which were inaccessible until now, allowing the use of gene manipulated mice to investigate, for example, the mechanisms involved in the development of diabetic microangiopathy.

Rab3a controls exocytosis in cholecystokinin-secreting cells.

The expression of rab3A and rab3D isoforms in the enteroendocrine, cholecystokinin-secreting, cell lines STC-1 and GLUTag is here demonstrated. In contrast, rab3B is undetectable in these two cell lines, and rab3C is only slightly expressed in GLUTag cells. Using a transient co-transfection system with human growth hormone as reporter protein, we show that overexpression of the GTPase-deficient mutant rab3AQ81L, but not rab3DQ81L, significantly decreases human growth hormone secretory responses to various agonists in STC-1 cells. These results indicate that endocrine cell lines of intestinal origin express rab3A and rab3D proteins, but the GTP-bound form of rab3A only acts as a negative modulator in the control of cholecystokinin secretion from STC-1 cells.

Insulin-like growth factor I receptors are expressed by the enteroendocrine cell line STC-1: relationship with proliferation and cholecystokinin expression.

Receptors for insulin-like growth factors (IGF-I and IGF-II) are expressed in mammalian intestinal epithelium. No information on the presence of IGF receptors in intestinal endocrine cells is available. We tested for IGF-I receptors the endocrine cell line STC-1, which synthesizes and processes cholecystokinin (CCK) among other peptides, and assessed the effects of IGF-I on cell growth and CCK content. Cell monolayers in serum-free culture medium specifically bound [125I]IGF-I. Scatchard analysis was consistent with a single class of high affinity binding sites (KD = 0.91 nM; Bmax = 4,700 sites/cell). In competitive binding assays, unlabeled IGF-I, IGF-II and insulin displaced in a dose-dependent manner [125I]IGF-I binding with the following potencies (KI): IGF-I (0.74 nM) > IGF-II (3 nM) >> insulin (1 microM). Affinity cross-linking with [125I]IGF-I using disuccinimidyl suberate and SDS-PAGE under reducing conditions yielded a polypeptide band with apparent Mr 130,000, consistent with the alpha-subunit of the IGF-I receptor. IGF-I and IGF-II (0.3-30 nM) dose-dependently stimulated [3H]thymidine incorporation, with a maximal response of 110% above basal. IGF-II was approximately 10-fold less potent than IGF-I, suggesting a mediation through IGF-I receptors. In addition, the numbers of cells treated with 3 nM IGF-I amounted to 116, 130 and 159% of control values after 1, 2 and 4 days of incubation, respectively (p < 0.05). A significant increase in the cell CCK contents was observed after a 48-hour exposure to 3 or 30 nM IGF-I. These results demonstrate IGF-I receptor expression by the enteroendocrine cell line STC-1. IGF-I stimulates proliferation in short-term experiments, and increases intracellular levels of CCK.

Galanin inhibits glucagon-like peptide-1 secretion through pertussis toxin-sensitive G protein and ATP-dependent potassium channels in rat ileal L-cells.

The neuropeptide galanin is widely distributed in the gastrointestinal tract and exerts several inhibitory effects, especially on intestinal motility and on insulin release from pancreatic beta-cells. The presence of galanin fibres not only in the myenteric and submucosal plexus but also in the mucosa, prompted us to investigate the regulatory role of galanin, and its mechanism of action, on the secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). Rat ileal cells were dispersed through mechanical vibration followed by moderate exposure to hyaluronidase, DNase I and EDTA, and enriched for L-cells by counterflow elutriation. A 6- to 7-fold enrichment in GLP-1 cell content was registered after elutriation, as compared with the crude cell preparation (929 +/- 81 vs 138 +/- 14 fmol/10(6) cells). L-cells then accounted for 4-5% of the total cell population. Bombesin induced a time-(15-240 min) and dose- (0.1 nM-1 microM) dependent release of GLP-1. Glucose-dependent insulinotropic peptide (GIP, 100 nM), forskolin (10 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA, 1 microM) each stimulated GLP-1 secretion over a 1-h incubation period. Galanin (0.01-100 nM) induced a dose-dependent inhibition of bombesin- and of GIP-stimulated GLP-1 release (mean inhibition of 90% with 100 nM galanin). Galanin also dose-dependently inhibited forskolin-induced GLP-1 secretion (74% of inhibition with 100 nM galanin), but not TPA-stimulated hormone release. Pretreatment of cells with 200 ng/ml pertussis toxin for 3 h, or incubation with the ATP-sensitive K+ channel blocker disopyramide (200 microM), prevented the inhibition by galanin of bombesin- and GIP-stimulated GLP-1 secretion. These studies indicate that intestinal secretion of GLP-1 is negatively controlled by galanin, that acts through receptors coupled to pertussis toxin-sensitive G protein and involves ATP-dependent K+ channels.