S.M. Kanse

Identification and characterization of glucagon-like peptide-1 7−36 amide-binding sites in the rat brain and lung

High‐affinity binding sites for glucagon‐like peptide‐1 7–36 amide (GLP‐1 7–36 NH2) were identified in rat brain and lung membranes. Binding of [125]GLP‐1 7–36 NH2 was rapid, reversible, specific, saturable and pH dependent. Specific binding in the central nervous system was particularly high in the hypothalamus and the brain stem. Oxyntomodulin, glucagon‐like peptide‐1, glucagon‐like peptide‐2 and glucagon were 100–1000‐fold less potent than GLP‐1 7–36 NH2 in competition for this binding site.

Endothelin in the gastrointestinal tract: Presence of endothelinlike immunoreactivity, endothelin-1 messenger RNA, endothelin receptors, and pharmacological effect

The possible production and role of endothelin in the gastrointestinal tract was investigated in rats by radioimmunoassay, Northern-blot hybridization, receptor assay using membrane preparations, and pharmacological study using gut strips. Endothelinlike immunoreactivity was detected in all regions (from stomach to colon) of the rat gastrointestinal tract (13-48 fmol/g wet tissue) including the mucosal layer of the ileum and colon (8.4 +/- 2.0 fmol/g wet tissue and 18.4 +/- 2.1 fmol/g wet tissue, respectively, mean +/- SEM; n = 5). Fast protein liquid chromatographic analysis of the endothelinlike immunoreactivity in jejunum, ileum, colon, and colon mucosa extracts showed peaks in the positions of endothelin-1 and endothelin-3. The presence of endothelin-1 messenger RNA was demonstrated by Northern-blot hybridization in the whole colon and pooled ileal and colonic mucosa, but not in the whole jejunum. Specific binding in the rat gastrointestinal tract was particularly high in the fundus of stomach, jejunum, ileum, and colon. In the ileum, many binding sites were found in the circular and longitudinal muscle layers, but few in the mucosal layer. Endothelin-1 and endothelin-3 caused contraction of rat stomach strips, rat colon, and guinea pig ileum. These findings indicate that endothelin is present in the rat gastrointestinal tract, perhaps produced by both vascular endothelial cells and mucosal epithelial cells, and can cause contraction of gastrointestinal smooth muscle. Thus, endothelin may have a physiological role in the control of gastrointestinal function.

Characterization of glucagon-like peptide-1-(7-36)amide in the hypothalamus.

The distribution of glucagon-like peptide-1-(7-36)amide like immunoreactivity (GLP-1-(7-36)NH2 IR) in rat brain was determined. Highest concentrations were found in the hypothalamus. A combination of gel chromatography and anion exchange chromatography showed that the majority of hypothalamic immunoreactivity exactly corresponded in position to synthetic GLP-1-(7-36)NH2. Chromatographic analyses of rat ileum demonstrated a similar pattern, whereas in rat pancreas mainly a large proglucagon fragment and GLP-1 were indicated. Determination of the subcellular distribution by differential centrifugation of hypothalamic tissue revealed that most of the GLP-1-(7-36)NH2 IR was present in the synaptosome fraction. GLP-1-(7-36)NH2 was released from hypothalamic tissue slices in a calcium-dependent fashion by potassium-induced depolarization. Thus GLP-1-(7-36)NH2 appears to fulfil two criteria for a neurotransmitter. No change was found in its hypothalamic content in streptozocin-induced diabetic rats compared to normal controls but a decrease was seen in hyperinsulinemic hyperglycemic KKAy mice compared to KK mice.

Production of Endothelin 1 by Cultured Bovine Retinal Endothelial Cells and Presence of Endothelin Receptors on Associated Pericytes

Endothelinlike immunoreactivity was detected by radioimmunoassay in medium conditioned by cultured endothelial cells obtained from bovine retinal microvessels (9.2 ± 6.5 pM, n = 4). Sephadex G-25 column chromatography and fast-protein liquid chromatography revealed that most of the endothelinlike immunoreactivity was eluted in an identical position to synthetic endothelin 1. Retinal capillary pericyte-conditioned medium contained 2.9 pM endothelinlike immunoreactivity. In contrast to endothelial cells, retinal pericytes were found to bind endothelin. The dissociation constant and binding capacity were 0.14 nM and 1.5 × 105 sites/cell (n = 3), respectively. These findings suggest that endothelin produced by the retinal endothelial cells binds to the pericytes, adding support to the suggestion that pericytes in the retina may have a musclelike function.

Cytokine stimulated endothelin release from endothelial cells

Endothelin release from bovine endothelial cells of the aorta, pulmonary artery, and retinal microvessels was measured in response to various cytokines. Transforming growth factor beta (0.05-5 ng/ml) was found to be a potent stimulator (3-4 fold increase) of endothelin secretion in all three cell types. Tumour necrosis factor alpha (0.1-10 ng/ml) and interferon gamma (8-800 U/ml) had a small (1.5-2 fold increase) but significant effect on endothelin secretion from endothelial cells of large vessels but not the retinal microvessels. Interleukin-1 beta, Interleukin-6 and interleukin-8 at various doses did not affect endothelin secretion. These effects were observed at various time points from 6-24 hrs and indicate that of the cytokines tested, only transforming growth factor beta has a potent effect on endothelin release from endothelial cells of different organs.

Glucocorticoids induce endothelin release from vascular smooth muscle cells but not endothelial cells.

Vascular smooth muscle cells in culture are capable of secreting endothelin which is a vasoconstrictor and mitogenic peptide. The effect of glucocorticoids on endothelin release from vascular smooth muscle cells of the rat and rabbit aortas was investigated. Micromolar concentrations of dexamethasone and cortisol caused a 2 to 5-fold increase in endothelin release from the two smooth muscle cell types but no such response was observed in endothelial cells of the bovine aorta. Glucocorticoids appear to selectively induce endothelin release from vascular smooth muscle cells and this may be relevant to glucocorticoid-induced hypertension.

Production of endothelin by vascular smooth muscle cells.

Endothelin (ET) production by cultured vascular smooth muscle cells isolated from rat and rabbit aortae was measured by a specific radioimmunoassay. Vascular smooth muscle cells released ET at a rate of 0.6 (range of 0.1-1) fmol/10(5) cells/24 h compared to 45 (range of 10-80) fmol/10(5) cells/h for endothelial cells. ET immunoreactivity was confirmed as ET-1 by fast protein liquid chromatography. Calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) suppressed basal release of ET by 24-66% from both rat and rabbit vascular smooth muscle. Forskolin and dibutryl-cAMP similarly suppressed ET release by 33-86%, suggesting that increased intracellular cAMP may account for the mechanism of action of CGRP and VIP. In contrast, CGRP and VIP did not have any effect on ET release from endothelial cells. The characteristics and regulation of ET binding sites on vascular smooth muscle cells were measured using [125I]ET-1 as the radioligand. Pretreatment with CGRP, VIP, forskolin, and dibutryl-cAMP increased the ET receptor density on both smooth muscle cell types. The low level production of ET-1 by vascular smooth muscle cells may have an important autocrine function and may be under regulation of neuropeptides localized to perivascular nerves.

Isolation and characterisation of GLP‐1 7–36 amide from rat intestine Elevated levels in diabetic rats

Glucagon‐like peptide‐1 (GLP‐1) was purified to homogeneity by HPLC and anion‐exchange chromatography. A molecular mass of 3297.4 Da was obtained by FAB mass spectrometry which corresponded exactly to GLP‐1 7–36 NH2, providing evidence that amidation occurs at an arginine residue during the post‐translational processing of GLP‐1. The distribution of GLP‐1 7–36 NH2‐like immunoreactivity (GLP‐1 7–36 NH2 IR) was determined in the rat gastrointestinal tract. Highest concentrations were found in terminal ileum and colon. Streptozocin‐induced diabetic rats, who showed a significant increase in food intake, had a significant increase of GLP‐1 7–36 NH2 IR in the colon.

Presence of immunoreactive endothelin in human saliva and rat parotid gland

The presence of immunoreactive endothelin (IR-ET) in human saliva and rat parotid gland was investigated by radioimmunoassay. The IR-ET concentration (mean +/- SEM) in saliva taken from normal volunteers was 2.0 +/- 0.2 pmol/l (n = 15). The IR-ET concentration in rat parotid gland was 19.2 +/- 2.2 fmol/g wet weight (n = 10). Fast protein liquid chromatography (FPLC) of human saliva extract revealed 6 peaks; one peak eluting in the void volume, one in a position between ET-1 and -3, and the other four in the positions of synthetic ET-1, -2, -3 and big ET(1-38), respectively. A similar pattern of rat parotid gland extract was noted with FPLC, except that there was no peak after the void volume. Presence of endothelin, a potent growth factor, in saliva and salivary gland points to a role in maintaining the integrity of the oral and gastrointestinal tract mucosa.

A study of the effects of human blood derivatives and individual growth factors on [3H]thymidine uptake in bovine retinal pericytes and endothelial cells

Pericytes disappear early, selectively and specifically from retinal capillaries in diabetic microangiopathy, but little is known of their growth and turnover in health and disease. We have studied the effects of human blood derivatives and of a panel of individual growth factors on [3H]thymidine incorporation in bovine retinal pericytes and endothelial cells. Human serum and platelet-rich plasma stimulated incorporation of the nucleotide in a dose-dependent manner in both cell types, and did so more potently than platelet-free plasma. Consistent and significant stimulation of DNA synthesis in pericytes was observed with basic fibroblast growth factor (ED50= 1.8×10−13 mol/l), acidic fibroblast growth factor (7.4× 10−12 mol/l), insulin-like growth factor 1 (8.6×10−10 mol/l), insulin (158 μU/ml) and endothelin-1 (6.1×10−10 mol/l). Transforming growth factor β1 inhibited DNA synthesis (ID50=3.6×10−10 mol/l) and so did heparin (1.4×10−6 mol/l) and low molecular weight heparin (2.9×10−6 mol/l). Retinal endothelial cells were stimulated by basic fibroblast growth factor (3.2×10−13 mol/l) and acidic fibroblast growth factor (1.3×10−9 mol/l), and inhibited by transforming growth factor β1, (1.6×10−12 mol/l). Neither cell type was stimulated by platelet-derived growth factor (A+B chain heterodimer), epidermal growth factor, growth hormone, or nerve growth factor (7S complex). The characteristics and active concentrations of the above growth factors suggest that none is solely responsible for the pericyte mitogenic activity of platelets, serum or plasma. Some, though, may play a role in the regulation of pericyte turnover through paracrine mechanisms which should be further investigated.