Somatostatin coupling to adenylyl cyclase activity in the mouse retina

The peptide somatostatin-14 (SRIF) acts in the mammalian retina through its distinct receptors (sst_1–5). Scarce information is available on SRIF function in the retina, including the elucidation of transduction pathways mediating SRIF action. We have investigated SRIF and SRIF receptor modulation of adenylyl cyclase (AC) activity in both wild-type (WT) retinas and sst₁ or sst₂ knock-out (KO) retinas, which are known to over-express sst₂ or sst₁ receptors respectively. In WT retinas, application of SRIF compounds does not affect forskolin-stimulated AC activity. In contrast, activation of sst₁ or sst₂ receptors inhibits AC in the presence of sst₂ or sst₁ receptor antagonists respectively. Results from sst₁ KO retinas demonstrate that either SRIF or the sst₂ receptor preferring agonist octreotide, pertussis toxin-dependently inhibit AC activity. In contrast, in sst₂ KO retinas, neither SRIF nor CH-275, an sst₁ receptor agonist, are found to influence AC activity. As revealed by immunoblotting experiments, in sst₁ KO retinas, levels of G_o proteins are 60% higher than in WT retinas and this increase in G_o protein levels is concomitant with an increase in sst_2A receptor expression. We conclude that interactions between sst₁ and sst₂ receptors may prevent SRIF effects on AC activity. In addition, we suggest that the density of sst₂ receptors and/or G_o proteins may represent the rate-limiting factor for the sst₂ receptor-mediated inhibition of AC.     

Rat hippocampal somatostatin sst3 and sst4 receptors mediate anticonvulsive effects in vivo: indications of functional interactions with sst2 receptors

Somatostatin-14 (SRIF) is a potent anticonvulsant in rodent models of limbic seizures in which the hippocampus is its major site of action. However, the distribution of hippocampal sst receptors and their role in the anticonvulsant effects of SRIF remain controversial. Moreover, striking differences have been described between mice and rats. In rats, sst₂ but not sst₁ receptors play a critical role in the anticonvulsant effects of SRIF. At present, the role of rat sst₃ and sst₄ receptors in these anticonvulsive effects remains unknown. Here we demonstrate in vivo anticonvulsive actions of rat hippocampal sst₃ and sst₄ receptors. Using microdialysis and telemetry-based electroencephalographic recordings we show that intrahippocampal administration of the sst₂ agonist L-779,976 (500 nM), the sst₃ agonist L-796,778 (100 nM) or the sst₄ agonist L-803,087 (100 nM) protects rats against focal pilocarpine-induced seizures. SRIF (1 μM)-, sst₃- and sst₄-mediated anticonvulsive actions are reversed by the selective sst₂ receptor antagonist cyanamid 154806 (100 nM). Moreover, the selective sst₃ antagonist SST3-ODN-8 (100 nM) blocks the sst₄-mediated anticonvulsant effect. Sst₃ antagonism does not reverse the sst₂- or SRIF-mediated anticonvulsant effects. Our findings provide the first in vivo evidence for potent anticonvulsive properties of sst₃ and sst₄ receptors in the rat hippocampus. Nevertheless, selective sst₂ receptor antagonism prevented these sst₃- or sst₄ receptor-mediated anticonvulsant effects, suggesting a functional cooperation with rat hippocampal sst₂ receptors.     

Protective role of somatostatin receptor 2 against retinal degeneration in response to hypoxia

In mouse retinal explants, octreotide, a somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor 2 (sst₂) agonist, prevents the hypoxia-induced vascular endothelial growth factor upregulation. In mice with oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity, either sst₂ overexpression or octreotide have been found to limit hypoxia-induced angiogenic processes. Here, we investigated whether sst₂ influences retinal degeneration in response to hypoxia in wild-type (WT), sst₁- and sst₂-knockout (KO) mice. In retinal explants, we determined the role of sst₂ on apoptotic signals. In control condition, caspase-3 activity and the Bax/Bcl-2 ratio were lower in sst₁-KO than in WT, but higher in sst₂-KO than in WT retinas. In all strains, a comparable increase in caspase-3 activity and the Bax/Bcl-2 ratio was observed after hypoxia. The hypoxia-induced increase in apoptotic signals was recovered by octreotide in both WT and sst₁-KO retinas. To investigate the role of sst₂ on retinal function, we recorded electroretinogram (ERG) in response to light flashes in OIR mice. ERG responses did not differ between WT and KO mice with the exception of oscillatory potentials (OPs) which, in sst₁-KO mice, displayed much larger amplitude. In all strains, hypoxia drastically reduced a-, b-waves and OPs. In both WT and sst₁-KO mice, octreotide recovered a- and b-waves, but did not recover OPs in sst₁-KO mice. Neither apoptotic signals nor ERG was affected by octreotide in sst₂-KO mice. These results show that sst₂ may protect retinal cells from hypoxia, thus implementing the background to establish potential pharmacological targets based on sst₂ pharmacology.     

Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst_2(b) receptor (CHOsst_2(b)) and compared these to their effects in cells expressing the sst_2(a) receptor (CHOsst_2(a)). In contrast to the sst_2(a) receptor, the sst_2(b) receptor did not mediate inhibition of bFGF (10 ng ml⁻¹)-stimulated re-growth and cell proliferation. Rather, SRIF (0.1–1000 nM) and angiopeptin (0.1–1000 nM) stimulated basal re-growth and proliferation of CHOsst_2(b) cells in a concentration-dependent manner (estimated pEC₅₀ values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst₂ receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative effect via the sst_2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst_2(a) and sst_2(b) receptor splice variants mediate opposite effects on cell proliferation.     

Pharmacological characterisation of human cerebral cortex somatostatin SRIF1 and SRIF2 receptors

Radioligand binding studies were performed in membranes of human cerebral cortex using [¹²⁵I]Tyr³-octreotide in the presence of 5 mM MgCl₂, [¹²⁵I]SRIF-14 ([¹²⁵I]Tyr¹¹-SRIF-14) and [¹²⁵I]CGP 23996 ([¹²⁵I]c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) both in the presence of 120 mM NaCl, to characterise the nature of the somatostatin (SRIF) receptors. The pharmacological profile of human brain SRIF recognition sites was compared with that of recombinant human SRIF₁ (sst₂-sst₃-sst₅) or SRIF₂ receptors (sst₁-sst₄) and with that of native rat sst₁, sst₂ and sst₄ receptors. [¹²⁵I]Tyr³-octreotide labelled binding sites in human cerebral cortex: B _max = 238 ± 36 fmol/mg protein and pK_d = 9.73 ± 0.08. The pharmacological profile of [¹²⁵I]Tyr³-octreotide labelled sites correlated very significantly with that of recombinant human sst₂ receptors (r = 0.98) and much less with those of recombinant human sst₃ (r = 0.65) or sst₅ receptors (r = 0.72). The correlation between [¹²⁵I]Tyr³-octreotide binding to native sst₂ receptors in human and rat cerebral cortex was also highly significant (r = 0.97). [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding (performed in the presence of 120 mM NaCl) in the human cerebral cortex identified very similar populations of sites B _max = 44 ± 7 and 36 ± 5 fmol/mg protein and pK_d = 9.44 ± 0.08 and 9.48 ± 0.10, respectively. The pharmacological profiles of the sites labelled with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 correlated highly significantly with those of recombinant human sst₁ (r = 0.97–0.99) or sst₄ receptors (r = 0.91–0.94). Similarly, the correlations between [¹²⁵I]SRIF-14 or [¹²⁵I]CGP 23996 binding in human cortex and [¹²⁵I]SRIF-14 binding to native sst₁ sites in rat cerebral cortex were also highly significant (r = 0.97 and 0.94, respectively). Finally, the pharmacological profile of native rat lung sst₄ sites determined with [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) correlated with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding in human cortex; r = 0.91 and 0.87, respectively. The present data show that in human cerebral cortex, [¹²⁵I]Tyr³-octreotide labels SRIF₁ receptor sites which are best characterised as of the sst₂ type, whereas [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 (both in the presence of 120 mM NaCl), label sites which fit almost equally well with sst₁ or sst₄ receptors and therefore are best described as of the SRIF₂ type. Under the conditions used, there was no evidence that either of these ligands would label sst₃ or sst₅ receptors in human cerebral cortex. Received: 8 August 1996 / Accepted: 8 November 1996     

Differential effects of somatostatin and angiopeptin on cell proliferation

1. 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.
2. 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.
3. 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.
4. 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).
5. 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 pK_B=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 (pK_B<7).
6. 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 pK_B value of 7.70±0.12.
7. 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.

     

Somatostatin receptors mediating inhibition of basal and stimulated electrogenic ion transport in rat isolated distal colonic mucosa

The aim of this study was to examine the potencies of several recently identified selective somatostatin (SRIF)-receptor ligands as inhibitors of electrogenic ion transport in the rat distal colonic mucosa with the view to identifying the SRIF receptor type involved. Under basal conditions, cumulative administration of SRIF and SRIF2g decreased short circuit current (SCC), a measure of electrogenic ion transport, with EC₅₀ values of 4 nM and 9 nM respectively. The peptidase inhibitors, phosphoramidon (1 M) and amastatin (10 M), had no effect on the potencies of either SRIF or SRIF₂₈. The inhibitory action of SRIF on basal SCC was suppressed by piretanide and diphenylamine-2-carboxylate, compatible with the assumption that the Na⁺K⁺2Cl^– co-transporter and Cl^– channels, respectively, may be involved in this antisecretory action of SRIF. Tetrodotoxin (1 M) had no effect on the antisecretory action of SRIF, suggesting that the process was not neuronally mediated.All of the SRIF analogues examined, with the exception of BIM-23056, maximally inhibited basal SCC to a similar extent as SRIF. Seglitide and octreotide were both more potent antisecretory agents than SRIF (respective EC₅₀ values, 0.4 nM and 1.5 nM) suggesting that this effect was mediated by a receptor belonging to the SRIF₁ receptor group. The most distinguishing feature of the rank order of agonist potencies was the high potency of the selective sst₂ receptor ligand, BIM-23027 (EC₅₀, value 0.32 nM), the weaker potency exhibited by the selective sst₅ receptor ligand, L-362855 (EC₅₀ value 21 nM), and the lack of agonist activity displayed by the selective sst₃ receptor ligand, BIM-23056 (EC₅₀ value > 1000 nM). This profile is comparable with that observed in binding studies on the recombinant sst₂ receptor.Forskolin-stimulated secretion was suppressed by SRIF analogues with the rank order of agonist potencies BIM-23027 > SRIF > L-362855 > BIM-23056 which resembled that exibited under basal conditions. However, the absolute potencies of these agonists were lower (respective EC₅₀ values 2 nM, 14 nM, 38 nM and > 1000 nM) whilst the magnitude of inhibition was about three fold greater. BIM-23027 and SRIF (both 30 nM) also inhibited carbachol-stimulated increases in basal SCC by 60–70%, while a similar concentration of L-362855 inhibited these responses by 11 %. BIM-23056 (1 M) had no effect on carbachol-simulated secretion. Radioligand binding studies on rat colonic mucosal membranes using [¹²⁵I]-Tyr¹¹-SRIF suggested heterogeneity of SRIF binding sites. Thus, SRIF and SRIF₂₈ competed for binding (IC₅₀ values, 0.32 and 0.63 nM, respectively) with Hill slopes less than unity; while seglitide and BIM-23027 both maximally displaced only 30–40% of specific binding with apparent high affinity (respective pIC₅₀ values, 10.1 nM and 10.0).In conclusion, SRIF decreases basal as well as both cAMP and Ca²⁺-dependent Cl^– secretion in rat colonic mucosa. The rank order of agonist potencies suggests that receptors resembling the recombinant sst₂ receptor mediate inhibition of basal and forskolin-stimulated secretion. Radioligand binding studies suggest that BIM-23027 interacts with a sub-population of [¹²⁵I]Tyr¹¹-SRIF binding sites in rat colonic mucosal membranes which probably correspond to the receptors mediating the antisecretory effects described here.     

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells

1. The aim of the present study was to determine the effect 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 sst₅ receptors (CHOsst₅), following partial denudation of a confluent 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 μM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC₅₀ 8.0–8.6) of the stimulated regeneration induced by sub-maximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml⁻¹), platelet-derived growth factor-BB (PDGF, 5 ng ml⁻¹) or endothelin-1 (ET-1, 100 nM). SRIF (pIC₅₀ 8.8) also inhibited bFGF-induced regeneration of CHOsst₅ cells.
3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml⁻¹) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM–1 μM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 μg ml⁻¹).
4. The sst₅ receptor-selective agonist, L-362,855 (pIC₅₀ 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst₅ cells whilst the sst₂ receptor-selective agonist, BIM-23027 (pIC₅₀ 6.8), was approximately 20 times weaker than SRIF.
5. The sst₅ receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst₅ cells (estimated pK_B values 8.8 and 8.3, respectively).
6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst₅ cells was abolished by pretreating cells with pertussis toxin (100 ng ml⁻¹) for 20 h.
7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst₅ receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive G_i/G_o proteins.

     

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

The somatostatin receptor subtype 5 in neuroendocrine tumours

Importance of the field: In recent years, scientific work has been intensified to unravel new (patho-) physiological insights, particularly regarding the functional role of somatostatin (SRIF) receptor subtype 5 (sst) and the development of novel sst₅-targeted SRIF analogues, in order to broaden medical therapeutic opportunities in patients suffering from neuroendocrine diseases.

Areas covered in this review: The scope of this review is primarily focused upon recent insights in sst₅-receptor physiology, novel sst₅-targeted treatment options predominantly directed towards pituitary adenomas, and gastroenteropancreatic neuroendocrine tumours.

What the reader will gain: An understanding of the potential that novel sst₅-targeted SRIF analogues might have in the medical treatment of Cushing’s disease and acromegaly, as demonstrated by translational research, based on pathophysiological data combined with results from clinical trials.

Take home message: The role of targeting sst₅ in gastroenteropancreatic neuroendocrine tumours remains to be established. The sst₅ subtype might function as sst₂ modulator in terms of receptor internalization and desensitization, and seems less important compared with sst₂-preferring SRIF analogues in the regulation of human insulin secretion by the pancreas. Finally, absence of sst₅ in corticotroph adenomas could be related to tumour aggressiveness in Cushing’s disease.     

Disulfiram deregulates HIF-α subunits and blunts tumor adaptation to hypoxia in hepatoma cells

Aim:

Disulfiram is an aldehyde dehydrogenase inhibitor that was used to treat alcoholism and showed anticancer activity, but its anticancer mechanism remains unclear. The aim of this study was to investigate the effects of disulfiram on the hypoxia-inducible factor (HIF)-driven tumor adaptation to hypoxia in vitro.

Methods:

Hep3B, Huh7 and HepG2 hepatoma cells were incubated under normoxic (20% O₂) or hypoxic (1% O₂) conditions for 16 h. The expression and activity of HIF-1α and HIF-2α proteins were evaluated using immunoblotting and luciferase reporter assay, respectively. Semi-quantitative RT-PCR was used to analyze HIF-mediated gene expression. Endothelial tubule formation assay was used to evaluate the anti-angiogenic effect.

Results:

Hypoxia caused marked expression of HIF-1α and HIF-1α in the 3 hepatoma cell lines, dramatically increased HIF activity and induced the expression of HIF downstream genes (EPO, CA9, VEGF-A and PDK1) in Hep3B cells. HIF-2α expression was positively correlated with the induction of hypoxic genes (CA9, VEGF-A and PDK1). Moreover, hypoxia markedly increased VEGF production and angiogenic potential of Hep3B cells. Disulfiram (0.3 to 2 μmol/L) inhibited hypoxia-induced gene expression and HIF activity in a dose-dependent manner. Disulfiram more effectively suppressed the viability of Hep3B cells under hypoxia, but it did not affect the cell cycle. Overexpression of HIF-2α in Hep3B cells reversed the inhibitory effects of disulfiram on hypoxia-induced gene expression and cell survival under hypoxia.

Conclusion:

Disulfiram deregulates the HIF-mediated hypoxic signaling pathway in hepatoma cells, which may contribute to its anticancer effect. Thus, disulfiram could be used to treat solid tumors that grow in a HIF-dependent manner.     

Somatostatin receptors in the ventral pallidum/substantia innominata modulate rat locomotor activity

Rational Somatostatin and its receptors (sst₁ and sst₂) have been localized in brain nuclei implicated in motor control, such as the nucleus accumbens, ventral pallidum (VP) and substantia innominata (SI). Objectives The objective of the study is to investigate the effect of somatostatin and selective sst₁ and sst₂ analogs infused in the VP/SI on the locomotor activity of the rat. Methods Somatostatin (15, 30, 60, 120 and 240 ng/0.5 μl/side), CH275 (sst₁ analog; 60, 180, 240 and 480 ng/0.5 μl/side), MK678 (sst₂ analog; 120, 240 and 480 ng/0.5 μl/side), L-809,087 (sst₄ agonist, 240 ng/0.5 μl/side) or saline (vehicle) were infused bilaterally in the VP/SI of the rat and locomotor activity measured for 60 min. The effect of SRA-880 (sst₁ antagonist) and CYN-154806 (sst₂ antagonist) on somatostatin-, CH275- and MK678-mediated locomotor activity was also ascertained. Results Somatostatin decreased locomotor activity in the first 30 min after its infusion in the VP/SI and in a dose-dependent manner. The sst₁ and sst₂ antagonists, SRA-880 and CYN-154806, respectively, reversed the somatostatin effect. The sst₁ and sst₂ agonists CH275 and MK678, respectively, mimicked somatostatin’s actions, while the selective sst₄ agonist L-809,087 had no effect. Moreover, SRA-880 and CYN-154806 reversed the respective agonist action on locomotor activity. Conclusion The present study provides functional evidence for the presence of sst₁ and sst₂ receptors in the VP/SI and their implication in motor control. The mechanism via which somatostatin and agonists mediate the attenuation of locomotor activity is presently being investigated.     

Somatostatin increases rat locomotor activity by activating sst2 and sst4 receptors in the striatum and via glutamatergic involvement

The involvement of striatal somatostatin receptors (sst₁, sst₂ and sst₄) in locomotor activity was investigated. Male Sprague–Dawley rats, 280–350 g, received in the striatum bilateral infusions of saline, somatostatin, and selective sst₁, sst₂, and sst₄ ligands. Spontaneous locomotor activity was recorded for 60 min. The involvement of excitatory amino acid receptors (AMPA and NMDA) on somatostatin’s actions was also examined. Western blot analysis was employed for the identification of somatostatin receptors in striatal membranes. Somatostatin, sst₂ and sst₄, but not sst₁, selective ligands increased rat locomotor activity in a dose-dependent manner. Blockade of AMPA and NMDA receptors reversed somatostatin’s actions. In conclusion, striatal somatostatin receptor activation differentially influence rat locomotor activity, while glutamatergic actions underlie the behavioral actions of somatostatin.     

Localization and pharmacological characterization of somatostatin sst2 sites in the rat cerebellum

Radioligand binding studies were performed in membranes of rat cerebellum using [¹²⁵I]-[Tyr³]octreotide ([¹²⁵I]204-090) to characterize the nature of cerebellar somatostatin receptors. Saturation experiments suggest the presence of a single class of binding sites with high affinity, pK_d = 9.53 ± 0.11, but low receptor density, B _max = 12.7 ± 1.0 fmol/mg protein. The pharmacological profile of [¹²⁵I]204-090 sites in cerebellar membranes was established using a range of ligands known to interact with SSTR-2 (now called sst₂) and other somatostatin (SRIF) receptors. SRIF analogues such as octreotide (SMS 201-995), seglitide (MK 678) and somatuline (BIM 23014) displayed very high affinity for cerebellar [¹²⁵I]204-090 binding sites. The data were compared to results obtained using the same ligand in rat cerebral cortex membranes known to represent sst₂ binding. The pharmacological characteristics of the cerebellar sites were in close correlation with those of the cerebral cortex (r = 0.976, n = 19, p < 0.001) and CHO-cells expressing human recombinant sst₂ receptor (r = 0.977, n = 19, p < 0.001). By contrast, there was very little correlation between cerebellar binding and published affinities for rat sst₅ receptors (r = 0.465), for which octreotide has also high affinity. In vitro autoradiographic studies performed in cerebellar slices using [¹²⁵I]204-090 demonstrated the presence of binding sites in the molecular layer of the rat cerebellum. In situ hybridization studies using sst₂ receptor mRNA selective oligoprobes confirmed the presence of sst₂ receptor mRNA in the rat cerebellum. Together, the present data demonstrate the presence of a low density of SRIF receptors in the molecular layer of the adult rat cerebellum which are best characterized as sst₂. This is the first pharmacological characterization and localization of sst₂ receptors in the adult rat cerebellum.     

Somatostatin receptor agonists and antagonists

Somatostatin is a cyclic peptide that is widely distributed in the CNS, the periphery and in a variety of tumours. Two biologically active forms, somatotropin release-inhibiting factor (SRIF)-14 and SRIF-28, exert their effects through activation of five G-protein-coupled receptor subtypes (sst₁ – sst₅). These peptides act as neurotransmitters or hormones and inhibit the secretion of other peptides, such as insulin, growth hormone and glucagon. Metabolically stable peptide and structurally diverse non-peptide analogues have been developed as subtype-selective agonists and antagonists. The availability of these novel SRIF analogues will greatly facilitate our understanding of the function and role of specific SRIF receptors. SRIF analogues offer therapeutic potential in the regulation of hormone secretion, diseases of the CNS and periphery and in the treatment and diagnosis of various tumours. This review will focus on an overview of SRIF, new developments related to SRIF role and function and the discovery of novel peptide and non-peptide agonists and antagonists.     

Ultravlolet-B induced expression of hypoxia-inducible factor 1α, transferrin receptor through EGFR/PI3K/AKT/DEC1 pathway

The aim of this research was to explore the effects and signaling pathway of ultraviolet-B (UVB) irradiation on the expression of hypoxia-inducible factor 1α (HIF-1α) and transferrin receptor (TfR). HIF-1α protein was measured by Western blot method. Expressions of epidermal growth factor receptor (EGFR), phosphor-EGF-R and TfR after UVB irradiation were determined with flow cytometry. After UVB irradiation, mRNA levels of HIF-1α and TfR were detected by real time-PCR. Results showed that compared with control groups, UVB was able to induce HIF1α and TfR protein expression in a dose-and time-dependent manner in HaCat cells (P < 0.05). TfR mRNA was expressed in a dose-dependent manner and reached a peak at the 8th hour in HaCat cells (P < 0.05) whereas HIF-1α mRNA expression was not affected by UVB treatment (P>0.05). The EGFR/PI3K/AKT signaling pathway was required for the induction of HIF-1α and TfR expression induced by UVB. UVB induced activation of EGFR in HaCat cells and EGFR regulated expression of TfR and HIF-1α. EGFR (−/−) MEF did not increase the HIF1 expression following UVB irradiation (P>0.05). In contrast, EGFR (+/+) MEF strongly enhanced HIF1α expression after UVB irradiation (P < 0.05). PD153035, a selective inhibitor of EGFR tyrosine kinase, inhibited the TfR protein expression in UVB-treated cells in a dose-dependent manner (P < 0.05). PI3K inhibitors, LY294002 and wortmannin, inhibited HIF-1α and TfR expressions induced by UVB (P < 0.05). The DEC1 (−/−) HaCat cells did not increase their TfR and HIF-1α expressions following UVB irradiation (P>0.05). In contrast, DEC1 (+/+) HaCat cells strongly enhanced TfR and HIF-1α protein expression after UVB irradiation (P < 0.05). We conclude that UVB induces TfR and HIF-1α expressions via EGFR/PI3K/AKT/DEC1 signaling pathway.