No abstract

The peptide hormone somatostatin exhibits antiproliferative activity by interacting with the G protein-coupled sst 2 or sst 5 receptor types. We show here that somatostatin at the human recombinant sst 4 receptor induced a concentration-dependent increase in proliferation (EC 50 20 nM) with a maximal response 5-fold greater than that produced by its synthetic analog, L-362,855. Analysis of the phosphorylation status of extracellular signal-regulated kinase (ERK)1 and ERK2 showed temporal differences in the changes evoked by the agonists. Phosphorylation induced by somatostatin (100 nM) peaked 10 min after the application and produced a response that continued for at least 4 h. In contrast, L-362,855 (1 M) showed transient phosphorylation that had declined to basal levels by 1 h. However, both agonists induced rapid and sustained tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) which was pertussis toxin-insensitive. Serine phosphorylation of STAT3 was only apparent after somatostatin treatment and was abolished by pertussis toxin or PD 98059, together with the associated increases in proliferation. Mitogen-activated protein/ERK kinase-1 inhibition also decreased the time interval over which somatostatin-induced ERK phosphorylation was observed (<2 h). We conclude that the difference in the magnitude of the proliferative response evoked by the two agonists at the sst 4 receptor can be accounted for by their differential ability to phosphorylate STAT3 on serine residues and supports the concept that selective signaling can be achieved through pharmacological diversity.Investigations of the growth effects of somatostatin on both normal and neoplastic human tissues suggest that it has a complex mechanism of action, inducing a direct antiproliferative response in a variety of cell types (1-3) in addition to reducing the circulatory levels of mitogenic hormones and growth factors (4, 5). Somatostatin transduces its direct action by stimulating G protein-coupled receptors, named sst 1-5 (6), although little is known as to the identity of the receptor types mediating its antiproliferative functions in tissues, and information has been largely restricted to studies utilizing partially selective receptor analogs (7). Numerous reports have demonstrated the expression of a high density of somatostatin receptors on a variety of human cancer cells (4, 8). The antiproliferative action of either somatostatin or its more metabolically stable analog octreotide, however, does not correlate with this expression, having inhibitory actions on pancreatic (9) and breast tumors (1) but eliciting no effect on the growth of small cell lung (10) and colon tumors (11). Growth-promoting effects of somatostatin have also been described in vitro on human pancreatic carcinoid (12) and epidermoid carcinoma cells (13), whereas in rat mesangial cells, somatostatin stimulates proliferation in the absence of serum but inhibits the growth of proliferating cells (14).As part of a study to resolve some of the appare...

1 The aim of the present study was to determine the e ect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst 5 receptors (CHOsst 5 ), following partial denudation of a con¯uent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2 In VSMC, SRIF (0.1 nM ± 1 mM) had no e ect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC 50 8.0 ± 8.6) of the stimulated regeneration induced by submaximal concentrations of basic ®broblast growth factor (bFGF, 10 ng ml 71 ), platelet-derived growth factor-BB (PDGF, 5 ng ml 71 ) or endothelin-1 (ET-1, 100 nM). SRIF (pIC 50 8.8) also inhibited bFGFinduced regeneration of CHOsst 5 cells. 3 In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml 71 ) was due to an anti-proliferative e ect, rather than an e ect on cell migration, as SRIF (0.1 nM ± 1 mM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 mg ml 71 ). 4 The sst 5 receptor-selective agonist, L-362,855 (pIC 50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst 5 cells whilst the sst 2 receptor-selective agonist, BIM-23027 (pIC 50 6.8), was approximately 20 times weaker than SRIF. 5 The sst 5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGFinduced regeneration in both VSMC and CHOsst 5 cells (estimated pK B values 8.8 and 8.3, respectively). 6 SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst 5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml 71 ) for 20 h. 7 These ®ndings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst 5 receptors. Furthermore this inhibitory e ect is transduced via pertussis toxin-sensitive G i /G o proteins.

Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst2/sst5 receptor‐selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO‐K1 cells stably transfected with the human or rat recombinant sst2 or sst5 receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors. In CHO‐K1 cells, expressing either human or rat recombinant sst2 or sst5 receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1–1000 nM) had no effect on basal re‐growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml−1) stimulated re‐growth of these cells. SRIF (0.1–1000 nM) caused a concentration‐dependent inhibition of the bFGF‐stimulated re‐growth in CHO‐K1 cells expressing human sst2 (h sst2) or sst5 (h sst5) receptors (pIC50=8.05±0.03 and 8.56±0.12, respectively). In contrast, angiopeptin (0.1–1000 nM) acted as a partial agonist at the h sst2 receptor (44.6±2.7% inhibition of the bFGF‐stimulated re‐growth at 100 nM; pIC50=8.69±0.25) but was devoid of any agonist activity at the h sst5 receptor. In CHO‐K1 cells stably expressing rat recombinant sst2 (r sst2) or sst5 (r sst5) receptors, SRIF (0.1–1000 nM) was able to inhibit the bFGF‐stimulated re‐growth (pIC50=7.98±24 and 8.50±0.12, respectively). Angiopeptin (0.1–1000 nM) caused a concentration‐dependent inhibition of bFGF‐stimulated re‐growth at the r sst2 receptor (pIC50=8.08±0.24) but acted as a partial agonist at the r sst5 receptor (maximum response=57.7±3.6% inhibition of bFGF‐stimulated re‐growth at 100 nM; pIC50=8.60±0.16). Although angiopeptin was inactive as an agonist at the h sst5 receptor, 100 nM angiopeptin potently antagonized the SRIF‐induced inhibition of proliferation in CHO h sst5 (estimated pKB=10.4±0.3). 5‐Hydroxytryptamine (0.1 nM–10 μM) also inhibited bFGF‐stimulated re‐growth (pIC50=8.36±0.11) and angiopeptin had no effect on this response (pKB<7). SRIF (0.1–1000 nM) caused a concentration‐dependent (pIC50=8.04±0.08) inhibition of bFGF‐stimulated re‐growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF‐stimulated re‐growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration‐effect curve to SRIF with an estimated pKB value of 7.70±0.12. These findings suggest that the low intrinsic activity of angiopeptin at the h sst2 receptor, combined with its lack of agonist activity at the h sst5 receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

The processes of wound repair were investigated using an in vitro model of mechanical injury on confluent cell monolayers of either human umbilical vein endothelial cells (HUVEC), aortic endothelial (RAEC) or smooth muscle cells (VSMC) of the rat. A mechanical wounder was used to produce 11 parallel (400 microm wide) lesions across the monolayer and the movement of cells into the denuded area was quantified using image analysis. The lesioned area recovered completely in 72h, with proliferation occurring after 24h for endothelial cells and 18h for VSMC, as detected by an increase in cell numbers. The cell migration inhibitor Taxol (1ng/ml) abolished the increase in repair of HUVEC monolayers in the first 24h of repair, while actinomycin D had no effect before 24h but thereafter abolished the further repair which was associated with increased cell numbers. Repair of endothelial cells was accelerated by basic fibroblast growth factor (bFGF), vascular endothelial growth factor or platelet-derived growth factor-BB (PDGF), and in VSMC both bFGF and PDGF increased repair. This simple in vitro model of mechanical injury allows a quantitative study of the repair processes of a previously confluent monolayer and thus is a representation of mechanical damage in vivo.