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Previous studies have shown antifibrotic effects of somatostatin. Since hepatic stellate cells (HSC) express somatostatin receptors and play a key role in hepatic fibrogenesis, we investigated the in vitro antifibrotic effect of somatostatin on rat HSC. At day 12 after isolation, cells were exposed to different concentrations of somatostatin (10(-6)-10(-9) mol l(-1)). mRNA expression of collagen types I and III, and of smooth muscle alpha-actin (alpha-SMA) was analysed by Northern blotting. At 10(-9) mol l(-1), somatostatin significantly reduced mRNA expression of collagen I (72.3 +/- 10.7%; 95% confidence interval (95% CI): 45.5-99.0), collagen III (79.0 +/- 4.5%; 95% CI: 67.6-90.4) and alpha-SMA (65.7 +/- 5.9%; 95% CI: 51.1-80.2), as compared to control normalized at 100%. These results were confirmed by quantitative RT-PCR. Cycloheximide experiments indicated that somatostatin has no direct transcriptional effect.Using immunoprecipitation, we demonstrated that somatostatin also decreased de novo synthesis of collagen I (73 +/-10%; 95% CI: 48-98%), collagen III (65 +/- 13%; 95% CI: 33-97%) and alpha-SMA (47 +/- 9%; 95% CI: 25-69%). Remarkably, at higher concentrations, somatostatin did not suppress collagen mRNA expression nor de novo protein synthesis. We ascribe this observation to desensitization of the cells for somatostatin. Cell proliferation, as measured by 5-bromo-2'-deoxyuridine labelling, was not altered by somatostatin. No significant effect on the intermediate and actin cytoskeleton were detected by immunohistochemistry and Western blotting. Our findings imply that in vivo antifibrotic effects of somatostatin could result partially from a direct action of somatostatin on HSC, but other, in vivo effects are probably also involved.

Functional studies indicate that the midbrain periaqueductal grey (PAG) is involved in the analgesic actions of somatostatin; however, the cellular actions of somatostatin in this brain region are unknown. In the present study, whole-cell patch clamp recordings were made from rat PAG neurons in vitro. In 93% of acutely isolated neurons, somatostatin inhibited Ca(2+)-channel currents. This effect was mimicked by the sst-2 selective agonist BIM-23027, but not by the sst-1 and sst-5 selective agonists CH-275 and L-362855. In brain slices, 81% of neurons responded to somatostatin (300 nm) with an increase in K(+) conductance that reversed polarity at -114 mV. A greater proportion of somatostatin-sensitive neurons (93%) than somatostatin-insensitive neurons (53%) responded to the opioid agonist met-enkephalin (10 microm). Somatostatin also reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). The actions of somatostatin in brain slices were mimicked by BIM-23027, but not by CH-275. Somatostatin had a variable effect on the rate of spontaneous miniature IPSCs in normal external potassium solutions. In high external potassium solutions, somatostatin reduced the rate of miniature IPSCs in all neurons, and this inhibition was abolished by addition of Cd(2+) (30 microm). Somatostatin had no effect on the amplitude of miniature IPSCs. These results indicate that somatostatin acts via sst-2 receptors to directly inhibit a subpopulation of PAG neurons by activating a potassium conductance and inhibits GABA release within PAG via a presynaptic Ca(2+)-dependent mechanism. Thus, like opioids, somatostatin has the potential to exert pre- and postsynaptic disinhibitory effects within the PAG.

We have investigated the regulation of hormone secretion from rat pancreatic islets by the GABAB receptors (GABABRs). Inclusion of the specific GABABR antagonist CGP 55845 in the extracellular medium increased glucose-stimulated insulin secretion 1.6-fold but did not affect the release of glucagon and somatostatin. Conversely, addition of the GABABR agonist baclofen inhibited glucose-stimulated insulin secretion by approximately 60%. Using RT-PCR, transcription of GABABR1a-c,f and GABABR2 subunits was detected in beta-cells. Measurements of membrane currents and cell capacitance were applied to single beta-cells to investigate the mechanisms by which GABABR activation inhibits insulin secretion. In perforated-patch measurements, baclofen inhibited exocytosis elicited by 500-ms voltage-clamp depolarizations to 0 mV by < or = 80% and voltage-gated Ca2+ entry by only approximately 30%. Both effects were concentration-dependent with IC50 values of approximately 2 microm. The inhibitory action of baclofen was abolished in the presence of CGP 55845. The ability of baclofen to suppress exocytosis was prevented by pre-treatment with pertussis toxin and by inclusion of GDPbetaS in the intracellular medium, and became irreversible in the presence of GTPgammaS as expected for a process involving inhibitory G-proteins (Gi/o-proteins). The inhibitory effect of baclofen resulted from activation of the serine/threonine protein phosphatase calcineurin and pre-treatment with cyclosporin A or intracellular application of calcineurin autoinhibitory peptide abolished the effect. Addition of baclofen had no effect on [Ca2+]i and electrical activity in glucose-stimulated beta-cells. These data indicate that GABA released from beta-cells functions as an autocrine inhibitor of insulin secretion in pancreatic islets and that the effect is principally due to direct suppression of exocytosis.

1 We characterized the effects of intravenous GABA and preferential GABAA (muscimol), GABAB (R-baclofen and SKF-97541) and GABAC agonists (imidazole-4-acetic acid) on gastric acid secretion in urethane-anesthetized mice implanted with a gastric cannula, and determined the role of vagal cholinergic mechanisms, and gastrin and somatostatin by using peptide immunoneutralization, the SSTR2 antagonist, PRL-2903, and SSTR2 knockout mice. 2 The selective GABA(B) agonists R-baclofen (0.1-3 mg kg(-1), i.v.) and SKF-97541 (0.01-0.3 mg kg(-1), i.v.) induced a dose-related stimulation of gastric acid secretion. SKF-97541 was about 10 times more potent than R-baclofen stimulating gastric acid secretion. Neither GABA (0.1-100 mg kg(-1), i.v.) nor muscimol (0.1-3 mg kg(-1)) nor imidazole-4-acetic acid (0.1-10 mg kg(-1)) affected basal gastric acid secretion. 3 Stimulatory effects of SKF-97541 (0.1 mg kg(-1), i.v.) were blocked by the selective GABAB antagonist, 2-hydroxysaclofen, cholinergic blockade with atropine, subdiaphragmatic vagotomy or gastrin immunoneutralization. 4 Somatostatin immunoneutralization or SSTR2 blockade with PRL-2903 enhanced the secretory response to SKF-97541 (0.1 mg kg(-1), i.v.) by 78 and 105%, respectively. 5 In SSTR2 knockout mice, SKF-97541 (0.1 mg kg(-1), i.v.) increased basal gastric acid secretion by 48%. Neither GABA nor muscimol nor imidazole-4-acetic acid modified basal gastric acid secretion in SSTR2 knockout mice. 6 These results indicate that, in mice, stimulation of GABAB receptors increases gastric acid secretion through vagal- and gastrin-dependent mechanisms. Somatostatin implication might be secondary to the release of gastrin and the increase in gastric luminal acidity.

Interneuronal networks in neocortex underlie feedforward and feedback inhibition and control the temporal organization of pyramidal cell activity. We previously found that lower layer neocortical interneurones can reach action potential threshold in response to the stimulation of a single presynaptic cell. To better understand this phenomenon and the circuit roles of lower layer neocortical interneurones, we combined two-photon calcium imaging with whole cell recordings and anatomical reconstructions of low threshold spiking (LTS) interneurones from mouse neocortex. In both visual and somatosensory cortex, LTS interneurones are somatostatin-positive, concentrated in layer 5 and possess dense axonal innervation to layer 1. Due to the LTS properties, these neurones operate in burst and tonic modes. In burst mode, dendritic T-type calcium channels boosted small synaptic inputs and triggered low threshold calcium spikes, while in tonic mode, sodium-based APs evoked smaller calcium influxes. In both modes, the entire dendritic tree of LTS interneurones behaved as a 'global' single spiking unit. This, together with the fact that synaptic inputs to layer 5 LTS cells are facilitating, and that their axons target the dendritic region of the pyramidal neurones where bursts are generated, make these neurones ideally suited to detect and control burst generation of individual lower layer pyramidal neurones.

1. Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein-coupled receptors (sst(1)-sst(5)) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst(2) receptor stably expressed in CHO-K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)-driven luciferase expression. 2. Intracellular calcium was measured using a fluorometric imaging plate reader II (FLIPR II). SRIF-14 rapidly and transiently increased intracellular calcium with a pEC(50) of 8.74+/-0.03 (n=52). At 5 h after FLIPR II measurements, luciferase expression was determined. SRIF-14 concentration-dependently increased luciferase expression (pEC(50)=9.06+/-0.03, n=52). 3. Natural and synthetic agonist/antagonist ligands for SRIF receptors were tested in the duplex assay. Correlation of agonist potencies and efficacies between the two responses were significant (r(2)=0.83 and 0.90, pEC(50) and E(max), respectively). 4. Pertussis toxin pretreatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%. 5. Thapsigargin pretreatment abolished the SRIF-14/octreotide-mediated intracellular calcium increase but had no effect on luciferase expression. 6. In conclusion, SRIF stimulates an increase in intracellular calcium and SRE-luciferase expression via human sst(2) receptors in CHO-K1 cells. The increase in luciferase is mediated via G(i)/G(o) while intracellular calcium increase is mediated by both G(i)/G(o) proteins and pertussis toxin-insensitive G proteins, and is mainly via release of calcium from intracellular stores. SRIF ligands display a similar recognition profile suggesting that the ligand/receptor/G protein/effector interaction is similar for the two parameters.

1. Studies in rats suggest that PACAP modulates gastric acid secretion through the release of both histamine and somatostatin. 2. We characterized the effects of exogenous PACAP on gastric acid secretion in urethane-anesthetized mice implanted with a gastric cannula and in conscious 2-h pylorus ligated mice, and determined the involvement of somatostatin and somatostatin receptor type 2 (SSTR2) by using somatostatin immunoneutralization, the SSTR2 antagonist, PRL-2903, and SSTR2 knockout mice. 3. Urethane-anesthetized wild-type mice had low basal acid secretion (0.10+/-0.01 micromol (10 min)(-1)) compared with SSTR2 knockout mice (0.93+/-0.07 micromol (10 min)(-1)). Somatostatin antibody and PRL-2903 increased basal secretion in wild-type mice but not in SSTR2 knockout animals. 4. In wild-type urethane-anesthetized mice, PACAP-38 (3-270 microg kg(-1) h(-1)) did not affect the low basal acid secretion, but inhibited the acid response to pentagastrin, histamine, and bethanechol. 5. In wild-type urethane-anesthetized mice pretreated with somatostatin antibody or PRL-2903 and in SSTR2 knockout mice, peripheral infusion of PACAP-38 or somatostatin-14 did not inhibit the increased basal gastric acid secretion. 6. In conscious wild-type mice, but not in SSTR2 knockout mice, PACAP-38 inhibited gastric acid secretion induced by 2-h pylorus ligation. The antisecretory effect of PACAP-38 was prevented by immunoneutralization of somatostatin. 7. These results indicate that, in mice, peripheral PACAP inhibits gastric acid secretion through the release of somatostatin and the activation of SSTR2 receptors. There is no evidence for stimulatory effects of PACAP on acid secretion in mice.

Rat stomach ECL cells release histamine in response to gastrin. Submucosal microinfusion of endothelin or adrenaline, known to cause vasoconstriction and gastric lesions, mobilized striking amounts of histamine. While the histamine response to gastrin is sustainable for hours, that to endothelin and adrenaline was characteristically short-lasting (1-2 h). The aims of this study were to identify the cellular source of histamine mobilized by endothelin and adrenaline, and examine the differences between the histamine-mobilizing effects of gastrin, and of endothelin and adrenaline. Endothelin, adrenaline or gastrin were administered by submucosal microinfusion. Gastric histamine mobilization was monitored by microdialysis. Local pretreatment with the H1-receptor antagonist mepyramine and the H2-receptor antagonist ranitidine did not prevent endothelin- or adrenaline-induced mucosal damage. Submucosal microinfusion of histamine did not cause damage. Acid blockade by ranitidine or omeprazole prevented the damage, suggesting that acid back diffusion contributes. Gastrin raised histidine decarboxylase (HDC) activity close to the probe, without affecting the histamine concentration. Endothelin and adrenaline lowered histamine by 50-70%, without activating HDC. Histamine mobilization declined upon repeated administration. Endothelin reduced the number of histamine-immunoreactive ECL cells locally, and reduced the number of secretory vesicles. Thus, unlike gastrin, endothelin (and adrenaline) is capable of exhausting ECL-cell histamine. Microinfusion of alpha-fluoromethylhistidine (known to deplete ECL cells but not mast cells of histamine) reduced the histamine-mobilizing effect of endothelin by 80%, while 1-week pretreatment with omeprazole enhanced it, supporting the involvement of ECL cells. Somatostatin or the prostanoid misoprostol inhibited gastrin-, but not endothelin-stimulated histamine release, suggesting that endothelin and gastrin mobilize histamine via different mechanisms. While gastrin effectively mobilized histamine from ECL cells in primary culture, endothelin had no effect, and adrenaline, a modest effect. Hence, the striking effects of endothelin and adrenaline on ECL cells in situ are probably indirect, possibly a consequence of ischemia.

1. We recently described the sensory nitrergic nature of the hepatic insulin sensitizing substance (HISS) mechanism linked to postprandial activation of anterior hepatic plexus fibres in rabbits. This study is designed to assess the involvement of the sensory pathways in this mechanism. 2. Selective sensory denervation of the anterior hepatic plexus (AHP) was achieved by a 3-day perineurial treatment with 2% capsaicin solution in Wistar rats (230-250 g). After 1 week, hyperinsulinaemic (100 micro U kg(-1)) euglycaemic (5.5 mmol kg(-1)) glucose clamp studies were performed to estimate insulin sensitivity. 3. The rats with regional AHP sensory denervation exhibited a significantly decreased insulin sensitivity, that is, 9.1+/-1.0 mg kg(-1) min(-1) glucose reinstalled euglycaemia vs 13.3+/-1.9 mg kg(-1) min(-1) glucose (P0.01) in control rats. 4. Acute partial hepatic denervation by AHP cut was without effect on insulin sensitivity, whereas chronic hepatic denervation induced insulin resistance was similar to that achieved by regional AHP capsaicin treatment. 5. Intraportal administration of L-NAME (10 mg kg(-1)) decreased, whereas capsaicin (0.3 mg kg(-1) min(-1)) increased insulin sensitivity. Neither atropine (1 mg kg(-1)) nor acetylcholine (1-10 micro g mg min(-1)) produced any significant effect. In animals with preceding regional capsaicin desensitization, none of the pharmacological manoeuvres modified the resulting insulin-resistant state. 6. Cysteamine (200 mg kg(-1) s.c.) is known to cause functional somatostatin depletion-induced insulin resistance similar to that produced by either chronic partial hepatic denervation or perineurial AHP capsaicin desensitization. Intraportal capsaicin (0.3 mg kg(-1) min(-1)) was unable to modify insulin resistance achieved by cysteamine. 7. We conclude that capsaicin-sensitive sensory fibres play a crucial role in neurogenic insulin sensitization known as the HISS mechanism without involvement of anatomical reflex-mediated circuits. The results also suggest that HISS is identical to somatostatin of AHP sensory neural origin.

1. The mouse corticotroph tumour cell line AtT-20 is a useful model to investigate the physiological role of native somatostatin (SRIF, Somatotropin release inhibitory factor) receptor subtypes (sst(1) - sst(5)). The objective of this study was to characterise the pharmacological features and the functional effects of SRIF receptors expressed by AtT-20 cells using radioligand binding and cAMP accumulation. 2. [(125)I]LTT-SRIF-28, [(125)I]CGP 23996, [(125)I]Tyr(10)-cortistatin-14 and [(125)I]Tyr(3)-octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (B(max)=315, 274, 239 and 206 fmol mg(-1), respectively). [(125)I]LTT-SRIF-28 labels significantly more sites than [(125)I]Tyr(10) -cortistatin-14 and [(125)I]Tyr(3) -octreotide as seen previously in cells expressing pure populations of sst(2) or sst(5) receptors. 3. SRIF analogues displaced the binding of the four radioligands. sst(2/5) receptor-selective ligands showed much higher affinity than sst(1/3/4) receptor-selective ligands. The binding profile of [(125)I]Tyr(3)-octreotide was different from that of [(125)I]LTT-SRIF-28, [(125)I]CGP 23996 and [(125)I]Tyr(10)-cortistatin-14. The sst(5/1) receptor-selective ligand L-817,818 identified two binding sites, one with subnanomolar affinity (sst(5) receptors) and one with micromolar affinity (sst(2) receptors); however, the proportions were different: 70 - 80% high affinity with [(125)I]LTT-SRIF-28, [(125)I]CGP 23996, [(125)I]Tyr(10)-cortistatin-14, but only 20% with [(125)I]Tyr(3)-octreotide. 4. SRIF analogues inhibited the forskolin-stimulated cAMP levels depending on concentration. sst(2/5) receptor-selective ligands were highly potent, whereas sst(1/3/4) receptor-selective ligands had no significant effects. The sst(2) receptor antagonist D-Tyr(8)-CYN 154806 competitively antagonised the effects of SRIF-14 and sst(2) receptor-preferring agonists, but not those of L-817,818. 5. The complex binding properties of SRIF receptor analogues indicate that sst(2) and sst(5) receptors are the predominant SRIF receptors expressed on AtT-20 cell membranes with no or only negligible presence of sst(1), sst(3) and sst(4) receptors. In the functional studies using cAMP accumulation, only sst(2) and sst(5) receptors appear to play a role. However, the "predominant" receptor appears to be the sst(2) receptor, although sst(5) receptors can also mediate the effect, when the ligand is not able to activate sst(2) receptors. This clearly adds flexibility to SRIF-mediated functional effects and suggests that the physiological role of SRIF and its analogues may be mediated preferentially via one subtype over another.

A 62-year-old woman on chronic haemodialysis since November 2008 for end-stage renal disease (ESRD) due to autosomal dominant polycystic kidney disease (ADPKD) had developed a massive polycystic liver (10.647 mL), causing severe abdominal discomfort, early satiety, dyspepsia and dyspnoea (Figure 1). On the basis of the demonstrated effectiveness of somatostatin analogues in reducing the liver volume in ADPKD patients [1, 2] and taking into account her reluctance for any liver surgery, we offered her a trial treatment with octreotide. We administered a single-test dose of short-acting subcutaneous 100 μg octreotide and observed the patient over a 24-h period. Diarrhoea, vomiting and abdominal cramps occurred several hours after administration, but disappeared the day after. We then started long-acting octreotide treatment at a monthly dose of 20 mg. A few hours after the injection, the patient suffered from diarrhoea, nausea and vomiting. While nausea and vomiting rapidly resolved, diarrhoea persisted over the whole next month. Thus, the monthly dose of octreotide was reduced to 10 mg. After 3 months of treatment, diarrhoea persisted with fatty and discoloured stools. At the same time, the patient began to report loss of nocturnal peripheral vision. She explained that, in the dark, her visual field was restricted, giving her the feeling of seeing ‘like in a tunnel’.

1. Transport of a fluorescent somatostatin analogue (NBD-octreotide) across freshly isolated functionally intact capillaries from porcine brain was visualized by confocal microscopy and quantitated by image analysis. 2. Luminal accumulation of NBD-octreotide showed all characteristics of specific and energy-dependent transport. Steady-state luminal fluorescence averaged 2 - 3 times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN. 3. The accumulation of NBD-octreotide in capillary lumens was inhibited in a concentration-dependent manner by unlabelled octreotide, by verapamil, PSC-833 and cyclosporin A, potent inhibitors of p-glycoprotein, and by leucotriene C(4), a strong modulator of Mrp2. Conversely, unlabelled octreotide reduced luminal accumulation of fluorescent BODIPY-verapamil on p-glycoprotein and of fluorescein-methotrexate, on Mrp2. None of the inhibitors used significantly reduced cellular accumulation of the fluorescent substrates. 4. Together, the data are consistent with octreotide being transported across the luminal membrane of porcine brain capillaries by both P-gp and Mrp2, providing further evidence that both transporters contribute substantially to the active barrier function of this endothelium.

1. Somatostatin (6.11 nmol kg(-1) i.p.) inhibited neurogenic plasma extravasation evoked by 1% mustard oil and non-neurogenic oedema induced by 5% dextran in the rat skin. 2. Cyclic synthetic octapeptide (TT-248 and TT-250) and heptapeptide (TT-232) somatostatin analogues proved to be more effective in reducing neurogenic and non-neurogenic inflammatory reactions but octreotide had no influence on either neurogenic or non-neurogenic inflammation. 3. TT-232 administered i.p. or i.v. (1.06 - 42.40 nmol kg(-1)) inhibited in a dose-dependent manner the plasma extravasation evoked by mustard oil in the rat's paw. Neither diclofenac (15.78 - 315.60 micromol kg(-1)) nor the selective COX-2 inhibitor meloxicam (2.95 - 569.38 micromol kg(-1)) attenuated the mustard oil-induced neurogenic plasma extravasation. 4. TT-232, diclofenac and meloxicam dose-dependently diminished non-neurogenic dextran-oedema of the paw the ED(35) values were 1.73 nmol kg(-1) for TT-232 and 34.37 micromol kg(-1) for diclofenac. 5. TT-232 inhibited in the dose range of 1.06 - 21.21 nmol kg(-1) the bradykinin-induced plasma extravasation in the skin of the chronically denervated paw. 6. Mustard oil-induced cutaneous plasma extravasation was dose-dependently diminished by s.c. TT-232 1, 2, 4, 6 or 16 h after the treatment. TT-232 (2 x 106, 2 x 212 and 2 x 530 nmol kg(-1) per day s.c. for 18 days) caused dose-dependent inhibition of chronic Freund adjuvant-induced arthritis during the experimental period. 7. TT-232 (200 and 500 nM) inhibited the release of SP, CGRP and somatostatin from the rat isolated trachea induced by electrical field stimulation (40 V, 0.1 ms, 10 Hz, 120 s) or by capsaicin (10(-7) M), but did not influence the basal, non-stimulated peptide release. 8. It is concluded that somatostatin analogues without endocrine functions as TT-232 are promising compounds with a novel site of action for inhibition of non-neurogenic and neurogenic inflammatory processes.

1. We have used in vivo microdialysis in anaesthetized rats to investigate whether levels of striatal somatostatin (SRIF) can be increased in response to application of the ionotropic glutamate receptor agonists AMPA and NMDA. 2. Application of both AMPA and NMDA (10, 50, 100 and 500 microM) for 20 min periods produced concentration-dependent increases in the extracellular levels of SRIF. A 500 microM dose of each compound was shown to be the most potent concentration tested, increasing levels of SRIF by 32 fold (NMDA) and 35 fold (AMPA). At lower concentrations (10 microM) NMDA failed to evoke significant amounts of SRIF while AMPA increased levels of the peptide 2.3 fold. 3. Application of the respective receptor antagonists APV (NMDA receptor) and DNQX (AMPA receptor) abolished the abilities of the agonists to evoke release of SRIF. Interestingly DNQX abolished the ability of NMDA to evoke release of the peptide as well. 4. The ability of both AMPA and NMDA to evoke increases in the levels of extracellular SRIF further illustrates the reciprocal relationship that exists between SRIF and glutamate in the striatum which impacts particularly on dopaminergic functioning in this region.

1. The bovine Galpha(14) is a member of the G(q) subfamily of G proteins that can regulate phospholipase Cbeta isoforms but the extent to which Galpha(14) recognizes different receptor classes is not known. 2. Galpha(14) was cotransfected with a variety of receptors in COS-7 cells, and agonist-induced stimulation of phospholipase C was then measured. 3. Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Galpha(14) stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation. 4. Among the three opioid receptors (mu, delta, and kappa), only the delta receptor was capable of stimulating IP formation when coexpressed with Galpha(14) in COS-7 cells. 5. A panel of G(i)- and G(s)-linked receptors was screened for their ability to stimulate IP accumulation via Galpha(14). The adenosine A(1), complement C5a, dopamine D(1), D(2) and D(5), formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Galpha(14), while the alpha(2)- and beta(2)-adrenoceptors were able to do so. 6. Galpha(14)-mediated stimulation of phospholipase Cbeta was agonist dose-dependent. These data demonstrate that although Galpha(14) can interact with different classes of receptors, it is much less promiscuous than Galpha(15) or Galpha(16).

There is controversy as to whether somatostatin sst(4) receptors internalize. In this study, CHO-K1 cells expressing human sst(4) receptor (CHOsst(4) cells) cells internalized [(125)I]-[(11)Tyr]-SRIF in a time-dependent manner, reaching a steady state at 60 min (1.4+/-0.1x10(4) molecules internalized per cell). Internalization was blocked by hypertonic sucrose (0.5 M), ATP depletion or by decreasing the temperature to 4 degrees C. Internalization of [(125)I]-[(11)Tyr]-SRIF was also inhibited (pIC(50) values) by increasing concentrations of SRIF (7.74), L-362855 (6.27) and NNC-296100 (6.50) with pIC(50) values approximately 10 fold lower than those obtained for inhibition of [(125)I]-[(11)Tyr]-SRIF binding to membrane homogenates. Internalized ligand recycled rapidly to the extracellular media (t(1/2) 3.9+/-0.7 min) with only 6.8+/-0.6% of internalized radioactivity remaining in the cell after 45 min. Confocal microscopy of permeabilized, HA-epitope tagged CHOsst(4) cells labelled with a Cy-3 conjugated antibody revealed little internal immunostaining after SRIF (1 microM) treatment, consistent with the small proportion of receptors (3.5%) estimated to be internalized by radioimmunoassay. In summary, CHOsst(4) cells internalized [(125)I]-[(11)Tyr]-SRIF in a clathrin- and ATP-dependent manner with subsequent rapid recycling to the extracellular medium. Rapid receptor recycling and the consequent low proportion of receptors internalized at any one time may explain the inability to visualize internalized receptors by confocal microscopy. It seems unlikely therefore that the marked receptor desensitization observed in CHOsst(4) cells following SRIF treatment can be accounted for by a decrease in cell surface receptor expression.

OBJECTIVES—Recent data indicate that alterations in brain neuropeptides may play a pathogenic role in dementia. Neuropeptide Y (NPY), somastostatin (SOM), and corticotropin releasing factor (CRF) are neuropeptides involved in cognitive performance. Decreased SOM and NPY concentrations have been found in patients with normal pressure hydrocephalus and are probably the result of neuronal dysfunction, which could potentially be restored by shunting. The effects of shunt surgery on preoperative SOM, NPY, and CRF concentrations were studied. Any improvements in neuropeptide concentrations that could lead to clinically significant neuropsychological and functional changes were also investigated. METHODS—A prospective study was performed in 14 patients with normal pressure hydrocephalus syndrome with a duration of symptoms between 3 months and 12 years. Diagnosis was based on intracranial pressure (ICP) monitoring and CSF dynamics. Concentrations of SOM, NPY, and CRF in lumbar CSF were determined before shunting and again 6-9 months after surgery. A battery of neuropsychological tests and several rating functional scales were also given to patients before and after shunting. RESULTS—After shunting, SOM and CRF concentrations were significantly increased in all patients. Concentrations of NPY were increased in 12 of the 14 patients studied. The clinical condition of 13of the 14 patients was significantly improved 6 months after surgery. This improvement was more pronounced in gait disturbances and sphincter dysfunction than in cognitive impairment. No significant differences in any of the neuropsychological tests were seen for the group of patients as a whole despite the increased neuropeptide concentrations. CONCLUSIONS—Shunting can restore SOM, NPY, and CRF concentrations even in patients with longstanding normal pressure hydrocephalus. However, despite the biochemical and clinical improvement in some areas such as ambulation and daily life activities, cognitive performance did not significantly improve. The role of neuropeptides in the diagnosis and treatment of patients with normal pressure hydrocephalus syndrome is discussed.

1. Somatostatin and the stable octapeptide analogues, octreotide and angiopeptin, were examined for their ability to stimulate the release of tritium from [(3)H]-arachidonic acid pre-loaded CHO-K1 cells expressing human recombinant sst(2), sst(3) or sst(5) receptors. 2. Somatostatin stimulated tritium release (pEC(50)) through the sst(2) (7.8+/-0.1) and sst(5) (7.3+/-0.2), but not the sst(3) receptor. Octreotide behaved as a full (sst(2) receptor) or partial agonist (sst(5) receptor), whereas angiopeptin behaved as a weak partial agonist at both receptor types. 3. Maximum responses to somatostatin through both receptor types were significantly reduced by pertussis toxin, whereas pEC(50) estimates were unaffected. 4. Inhibition of MEK1 or Src, but not PKA, PI 3-kinases or tyrosine kinases, by reportedly selective inhibitors reduced sst(2)-mediated responses by somatostatin, but not angiopeptin. A selective inhibitor of PKC (Ro-31-8220) reduced both somatostatin and angiopeptin responses. 5. These data provide further evidence for partial agonist activity of synthetic peptides of somatostatin. Furthermore, the somatostatin receptor signalling mechanisms which mediate arachidonic acid mobilization appear to be multiple and complex.

1. Three human adenocarcinoma cell lines, Colony-24 (Col-24), Col-6 and Col-1 have been studied as confluent epithelial layers able to transport ions vectorially in response to basolateral vasoactive intestinal polypeptide (VIP) and pancreatic polypeptides (PP). 2. Different species PP stimulated responses in Col-24 with Y(4)-like pharmacology. Bovine (b)PP, human (h)PP and porcine (p)PP were equipotent (EC(50) values 3.0--5.0 nM) while rat (r)PP, avian (a)PP and [Leu(31), Pro(34)]PYY (Pro(34)PYY) were significantly less potent. PYY was inactive. The PP pharmacology in Col-1 was comparable with Col-24. However, Col-6 cells were different; pPP had an EC(50) intermediate (22.0 nM) between that of bPP (3.0 nM) and hPP (173.2 nM), with aPP and rPP being at least a further fold less potent. 3. Deamidation of Tyr(36) in bPP (by O-methylation or hydroxylation) or removal of the residue resulted in significant loss of activity in Col-24. 4. GR231118 (1 microM) had no PP-like effects. In Col-24 and Col-1, GR231118 significantly attenuated bPP (30 nM) or hPP (100 nM) responses, but it did not alter bPP responses in Col-6. BIBP3226 and GR231118 both inhibited Y(1)-mediated responses which were only present in Col-6. 5. RT--PCR analysis confirmed the presence of hY(4) receptor mRNA in Col-24 and Col-1 epithelia but a barely visible hY(4) product was observed in Col-6 and we suggest that an atypical Y(4) receptor is expressed in this cell line.

Orphanin FQ (OFQ, also known as nociceptin) has been proposed to oppose the antinociceptive effect of endogenous opioid peptides in the brain. We sought to determine whether, conversely, the endogenous opioid peptides counteract a pronociceptive action of OFQ. In testing this hypothesis, naloxone, a non-selective opioid receptor antagonist, was used to block the action of endogenous opioid peptides. We then examined whether OFQ would produce hyperalgesia in the absence of such an endogenous opioidergic tone. Neither naloxone (1 mg kg(-1); s.c.) nor OFQ (up to 30 nmol; i.c.v.) alone induced any significant change in mean hot plate latency. However, OFQ dose-dependently produced hyperalgesia in rats pretreated with naloxone, implying that OFQ can indeed produce hyperalgesia once an endogenous opioidergic tone is inhibited. In subsequent studies, we used subtype selective opioid receptor antagonists to determine which class of opioid receptor is involved in this response. The effect of naloxone was reproduced using the selective mu-opioid receptor antagonist CTOP (D-Phe-Cyc-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), but not by administration of the delta-opioid receptor antagonist, naltrindole (NTI) or the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). These results suggest that endogenous opioid peptides acting at the mu-, but not kappa- or delta-opioid receptor may be counteracting the hyperalgesic effect of OFQ in rats.

N-methyl-D-aspartate (NMDA) receptors exist on noradrenergic axon terminals and mediate enhancement of noradrenaline (NA) release. We here investigated modulation by somatostatin (SRIF, somatotropin release inhibiting factor) of the NMDA-induced release of NA using superfused hippocampal synaptosomes. The NMDA response was increased by SRIF-28 and SRIF-14, but not SRIF-28((1 - 14)), whereas the release of [(3)H]-NA elicited by alpha-amino-3-hydroxy-5-methylisoxazide-4-propionic acid (AMPA) was unaffected. SRIF-14 did not mimic glycine at the NMDA receptor but activated SRIF receptors sited on noradrenergic terminals. The SRIF-14 effect was blocked by pertussis toxin but mimicked by mastoparan, a G-protein activator. BIM-23056, but not Cyanamid 154806, antagonized the SRIF-14 effect. This effect was mimicked by L362855, a partial agonist at the sst(5) subtype, but not by the new selective sst(1) - sst(4) receptor agonists L797591, L779976, L796778 and L803087. Protein kinase C (PKC) inhibitors (H7, staurosporine, GF 209103X, cheleritrine and sphingosine) prevented the SRIF-14 effect, while phorbol 12-myristate 13-acetate enhanced the NMDA response. SRIF-14 permitted NMDA receptor activation in the presence of 1.2 mM Mg(2+) ions, both in hippocampal synaptosomes and slices. Blockade of inositol-1,4,5-trisphosphate (InsP(3)) receptors with heparin abolished the effect of SRIF-14. It is concluded that SRIF receptors, possibly of the sst(5) subtype, can exert a permissive role on NMDA receptors colocalized on hippocampal noradrenergic terminals: activation of sst(5) receptors is coupled to pertussis toxin-sensitive G proteins enhancing phosphoinositide metabolism with activation of InsP(3) receptors and PKC; NMDA receptor subunits might be phosphorylated with consequent removal of the Mg(2+) block in absence of depolarization.

1. In rat pituitary tumour cells (GC cells), spontaneous oscillations of the intracellular concentration of Ca2+ ([Ca2+]i) induce growth hormone (GH) secretion that is inhibited by octreotide, a somatostatin (SRIF) agonist which binds to SRIF subtype (sst) receptor 2. The effects of its functional activation on the control of [Ca2+]i were investigated using fluorimetric measurements of [Ca2+]i. 2. SRIF decreases the basal [Ca2+]i and the [Ca2+]i rise in response to forskolin (FSK) through the inhibition of L-type voltage-dependent Ca2+ channels. 3. Pretreatment with octreotide or with L-Tyr8++ Cyanamid 154806, a sst2 receptor antagonist, abolishes the SRIF-induced inhibition of [Ca2+]i. Octreotide is known to operate through agonist-induced desensitization, while the antagonist operates through receptor blockade. 4. sst1 and sst2 receptor-immunoreactivities (-IRs) are localized to cell membranes. sst2, but not sst1 receptor-IR, internalizes after cell exposure to octreotide. 5. SRIF-induced inhibition of basal [Ca2+]i or FSK-induced Ca2+ entry is blocked by pertussis toxin (PTX). 6. FSK-induced cyclic AMP accumulation is only partially decreased by SRIF or octreotide, indicating that sst2 receptors are coupled to intracellular pathways other than adenylyl cyclase (AC) inhibition. 7. In the presence of H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA), SRIF-induced inhibition of basal [Ca2+]i is still present, although reduced in amplitude. 8. SRIF inhibits [Ca2+]i by activating sst2 receptors. Inhibition of AC activity is only partly responsible for this effect, and other transduction pathways may be involved.

The therapeutic potential of the somatostatin analogue RC-160 having antiproliferative activity, is limited by its short serum half life. To overcome this limitation, fatty acids namely butanoic acid and myristic acid were conjugated to the N-terminal residue of RC-160. The lipophilized derivatives of RC-160 were synthesized, purified by reverse phase HPLC and characterized by ES-mass spectroscopy. The antiproliferative activity of lipophilized derivatives of RC-160 on the growth of MIA-PaCa2 (human pancreatic carcinoma), DU145 (human prostate carcinoma), ECV304 (human umbilical chord endothelioma), as well as their antiangiogenic activity was evaluated in vitro. The relative stability of myristoyl-RC-160 towards degradation by proteases and serum was also determined. Myristoyl-RC-160 exhibited significantly higher antiproliferative efficacy than RC-160, on the above cell lines (P0.01). Receptor binding assays, demonstrated that the affinity of RC-160 towards somatostatin receptors remains unaltered by myristoylation. Unlike RC-160, the myristoylated derivative was found to have significantly greater resistance to protease and serum degradation (P0.01). Myristoyl-RC-160 exhibited significantly greater antiproliferative activity on ECV304, than RC-160 (P0.01). Myristoyl RC-160 could also inhibit capillary tube formation more efficiently than RC-160 in a dose dependent manner, suggesting that it possessed enhanced antiangiogenic activity in vitro (P0.001). Lipophilization of RC-160 with long chain fatty acids like myristic acid endows it with improved antiproliferative and antiangiogenic activity, stability and therapeutic index. British Journal of Pharmacology (2000) 109, 101 - 109

1DMe, a neuropeptide FF (NPFF) analogue, has been shown to produce antinociception and to enhance morphine analgesia in rats after intrathecal administration. To determine whether 1DMe could correct hyperalgesia and restore morphine efficacy in mononeuropathic (MN) and diabetic (D) rats we examined the spinal effect of 1DMe in MN and D rats without and after spinal blockade of mu- and delta-opioid receptors with CTOP and naltrindole, respectively. The influence of 1DMe on morphine-induced antinociception was assessed in the two models using isobolographic analysis. Whereas 1DMe intrathecally injected (0.1, 1, 7.5 microg rat(-1)) was ineffective in normal (N) rats, it suppressed mechanical hyperalgesia (decrease in paw pressure-induced vocalisation thresholds) in both MN and D rats. This effect was completely cancelled by CTOP (10 microg rat(-1)) and naltrindole (1 microg rat(-1)) suggesting that it requires the simultaneous availability of mu- and delta-opioid receptors. The combinations of morphine: 1DMe (80.6:19.4% and 99.8:0.2%, in MN and D rats, respectively) followed by isobolographic analysis, showed a superadditive interaction, relative to the antinociceptive effect of single doses, in D rats only. In N rats, the combination of morphine: 1DMe (0.5 mg kg(-1), i.v.: 1 microg rat(-1), i.t., ineffective doses) resulted in a weak short-lasting antinociceptive effect. These results show a different efficacy of 1DMe according to the pain model used, suggesting that the pro-opioid effects of the NPFF in neuropathic pain are only weak, which should contribute to hyperalgesia and to the impaired efficacy of morphine.

1. The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO-K1) cells to adenylate cyclase was investigated using receptor selective ligands. 2. Forskolin (10 microM)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin-14 and a number of receptor-selective agonists with a rank order of agonist potency typical of the sst5 receptor. L-362,855 and BIM-23056 behaved as full agonists. At higher somatostatin-14 concentrations there was sub-maximal inhibition resulting in a bell-shaped concentration-effect relationship. Pertussis toxin (PTx; 100 ng ml(-1), 18 h) pre-treatment abolished agonist-mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations. 3. The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 microM somatostatin-14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. 4. Pre-treatment with cholera toxin (ChTx; 20 microg ml(-1), 18 h) markedly inhibited sst5 receptor-mediated increases in cyclic AMP formation in intact cells. Somatostatin-14-stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO-K1 membranes and was inhibited by the synthetic peptide Galphasacetyl-354-372-amide (100 microM) by 65.9+/-3.5%, implicating a Galphas protein involvement in this response. 5. Activation of Galphas proteins by somatostatin-14 could be demonstrated with [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding and subsequent immunoprecipitation of 35S labelled Galphas proteins with anti-Galphas serum. 6. These data show that the sst5 receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving Galphas protein activation.

1. The effect of a new type 2 selective somatostatin (SRIF) receptor antagonist (DC-41-33) on somatostatin-induced inhibition of pentagastrin-stimulated gastric acid secretion in conscious, chronic gastric fistula equipped rats was studied. 2. Infused intravenously, DC-41-33 dose-dependently inhibits SRIF-induced inhibition of pentagastrin-stimulated gastric acid secretion with an IC50 of 31.6+/-1.2 nmol kg(-1) versus 10 nmol kg(-1) SRIF and blocks the inhibitory effects of SRIF when simultaneously co-infused. Its effectiveness provides additional evidence that SRIF-inhibition of gastric acid release is a SRIF type 2 receptor-mediated process. 3. DC-41-33 is able to completely reverse the inhibitory effect of glucose-dependent insulinotropic polypeptides, GIP and GIP-(1-30)NH2, and glucagon-like polypeptide, GLP-1(7-36)NH2, on pentagastrin-stimulated gastric acid secretion thus confirming that they exert these effects through stimulation of endogenous SRIF release. 4. DC-41-33 only partially blocks potent amylin and adrenomedullin-induced inhibition of gastric acid secretion, therefore suggesting that somatostatin may not function as a primary mediator in the action of these peptides. 5. Our results indicate that DC-41-33, is a potent in vivo inhibitor of exogenous and endogenous SRIF in rats. It represents a new class of SRIF analogues which should eventually provide excellent tools for further evaluating the many physiological roles of SRIF and its five receptor subtypes.

The bHLH–PAS transcription factor SIM1 is expressed during the development of the hypothalamic–pituitary axis in three hypothalamic nuclei: the paraventricular nucleus (PVN), the anterior periventricular nucleus (aPV), and the supraoptic nucleus (SON). To investigateSim1 function in the hypothalamus, we produced mice carrying a null allele of Sim1 by gene targeting. Homozygous mutant mice die shortly after birth. Histological analysis shows that the PVN and the SON of these mice are hypocellular. At least five distinct types of secretory neurons, identified by the expression of oxytocin, vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin, are absent in the mutant PVN, aPV, and SON. Moreover, we show that SIM1 controls the development of these secretory neurons at the final stages of their differentiation. A subset of these neuronal lineages in the PVN/SON are also missing in mice bearing a mutation in the POU transcription factor BRN2. We provide evidence that, during development of the Sim1 mutant hypothalamus, the prospective PVN/SON region fails to express Brn2. Our results strongly indicate that SIM1 functions upstream to maintain Brn2 expression, which in turn directs the terminal differentiation of specific neuroendocrine lineages within the PVN/SON.

1. The operational characteristics of somatostatin (SRIF) sst4 receptors are poorly understood. In this study, we have characterized human recombinant sst4 receptors expressed in CHO cells (CHOsst4) by radioligand binding and microphysiometry. 2. Increasing concentrations SRIF or other SRIF receptor ligands inhibited specific [125I]-Tyr11-SRIF binding in CHOsst4 cell membranes with respective pIC50 values of SRIF (8.82), L-362855 (7.40), BIM-23027 (5.5) and MK-678 (5.5). 3. These ligands displayed agonist activity, producing concentration-dependent increases in rates of extracellular acidification (EAR) with pEC50 values of SRIF (9.6) and L-362855 (8.0), respectively. BIM-23027 and MK-678 were at least 1000 times weaker than SRIF. The SRIF maximum was about 40% of that observed with L-362855. 4. In the presence of SRIF (0.1-1 nM), concentration-effect curves to L-362855 were displaced to the right with a progressive reduction in the L-362855 maximum. 5. When cells were only exposed to a single maximally effective concentration of SRIF or L-362855, there was no difference in the magnitude of the agonist-induced increase in EAR. However, a second agonist challenge, 30 min later showed that responses to SRIF but not L-362855 were markedly desensitized. 6. When concentration-effect curves to SRIF and L-362855 were obtained by combining data from cells exposed to only a single agonist concentration, SRIF (pEC50 9.2) was approximately 20 times more potent than L-362855 (pEC50 8.0) but the maxima were the same. Responses to both SRIF and L-362855 were abolished by pertussis toxin. 7. SRIF and L-362855-induced increases in EAR were inhibited by N-ethyl isopropyl amiloride (10 microM) but were not modified by inhibitors of PKC (Go-6976), MAP kinase (PD-98059), tyrosine kinase (genistein) or tyrosine phosphatase (sodium orthovanadate). 8. The results suggest that SRIF-induced increases in EAR in CHOsst4 cells involved activation of the Na+/H+ antiporter and were mediated via Gi/Go G proteins. Responses to SRIF, but not L-362855, were subject to marked desensitization which may be a consequence of differential activation of receptor-effector coupling pathways.

1. Neurogenic plasma extravasation evoked by topical application of 1% vv(-1) mustard oil on the skin of the acutely denervated rat hindleg (primary reaction) inhibited the development of a subsequent oil-induced plasma extravasation induced in the skin of the contralateral hindleg by 49.3+/-7.06% (n=9) and in the conjunctival mucosa due to 0.1% wv(-1) capsaicin instillation by 33.5+/-10.05% (n=6). The primary reaction also inhibited the non-neurogenic hindpaw oedema evoked by s.c. injection of 5% wv(-1) dextran into the chronically denervated hindpaw by 48.0+/-4.6% (n= 5). 2. Capsaicin injection (100 microg ml(-1) in 50 microl, s.c.) into the acutely denervated hindleg caused 56.5+/-4.0% (n=5) inhibition in the intensity of plasma extravasation elicited by 1% vv(-1) mustard oil smearing on the contralateral side. After chronic denervation, subplantar injection of 5% wv(-1) dextran elicited a non-neurogenic inflammatory response with intensive tissue oedema without causing any systemic anti-inflammatory effect. Bilateral adrenalectomy did not inhibit the mustard oil-induced anti-inflammatory effect in the contralateral hindleg. 3. Pretreating the rats with polyclonal somatostatin antiserum (0.5 ml rat(-1), i.v.) or with the somatostatin depleting agent cysteamine (280 mg kg(-1), s.c.) prevented the inhibitory action of mustard oil-induced inflammation on subsequent neurogenic plasma extravasation and strongly diminished the inhibition of non-neurogenic oedema formation evoked by dextran. 4. Exogenous somatostatin (10 microg kg(-1), i.p.) caused a 30.3+/-8.3% (n=6) inhibition of plasma extravasation caused by mustard oil smearing on the acutely denervated hindleg and this inhibitory effect was abolished by somatostatin antiserum (0.5 ml rat(-1), i.v.). The plasma level of somatostatin-like immunoreactivity (SST-LI) increased by 40.03+/-6.8% (n= 6) 10 min after topical application of 1% vv(-1) mustard oil on the acutely denervated hindpaws compared to the paraffin oil treated control group. Chronic denervation of the hindlegs or cysteamine (280 mg kg(-1), s.c.) pretreatment prevented the mustard oil-induced elevation of SST-LI in plasma. 5. It is concluded that chemical excitation of the capsaicin-sensitive sensory receptors not only induces local neurogenic plasma extravasation but also inhibits the development of a subsequent inflammatory reaction at remote sites of the body in the rat. A role for somatostatin in this systemic anti-inflammatory effect is suggested.

1. In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. 2. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. 3. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. 4. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand.

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-KI 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 pKB= 10.4+/-0.3). 5-Hydroxytryptamine (0.1 nM-10 microM) also inhibited bFGF-stimulated re-growth (pIC50=8.36+/-0.11) and angiopeptin had no effect on this response (pKB7). 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 pKB 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.

1. A high density of receptors for somatostatin (SRIF) exists in the anterior cingulate cortex but their function is unknown. Whole-cell patch clamp recordings were made from visualized deep layer pyramidal cells of the rat anterior cingulate cortex contained in isolated brain slices to investigate the putative effects of SRIF and to identify the receptor subtype(s) involved. 2. SRIF (1-1000 nM) produced a concentration-dependent outward current which was associated with an increased membrane conductance, was sensitive to Ba2+ (300 microM - 1 mM), and was absent in the presence of a maximal concentration of the GABA(B) receptor agonist, baclofen (100 microM). These observations suggest the outward current was carried by K+ ions. 3. SRIF analogues also elicited outward currents with a rank potency order of (EC50, nM): octreotide (1.8)BIM-23027 (3.7)SRIF (20)=L-362,855 (20). BIM-23056 was without agonist or antagonist activity. Responses to L-362,855 were unlike those to the other agonists since they were sustained for the duration of the application. 4. The sst2 receptor antagonist, L-Tyr8Cyanamid 154806 (1 microM), had no effect alone but partially reversed responses to submaximal concentrations of SRIF (100 nM, 44+/-6% reversal) and L-362,855 (100 nM, 70+/-6% reversal) and fully reversed the response to BIM-23027 (10 nM). In contrast, L-Tyr8Cyanamid 154806 did not antagonize the response to baclofen (10 microM). 5. We conclude that SRIF activates a K+ conductance in anterior cingulate pyramidal neurones via an action predominantly at sst2 receptors.

1. The effect of antidromic stimulation of the sensory fibres of the sciatic nerve on inflammatory plasma extravasation in various tissues and on cutaneous vasodilatation elicited in distant parts of the body was investigated in rats pretreated with guanethidine (8 mg kg(-1), i.p.) and pipecuronium (200 microg kg(-1), i.v.). 2. Antidromic sciatic nerve stimulation with C-fibre strength (20 V, 0.5 ms) at 5 Hz for 5 min elicited neurogenic inflammation in the innervated area and inhibited by 50.3 +/- 4.67% the development of a subsequent plasma extravasation in response to similar stimulation of the contralateral sciatic nerve. Stimulation at 0.5 Hz for 1 h also evoked local plasma extravasation and inhibited the carrageenin-induced (1%, 100 microl s.c.) cutaneous inflammation by 38.5 +/- 10.0% in the contralateral paw. Excitation at 0.1 Hz for 4 h elicited no local plasma extravasation in the stimulated hindleg but still reduced the carrageenin-induced oedema by 52.1 +/- 9.7% in the paw on the contralateral side. 3. Plasma extravasation in the knee joint in response to carrageenin (2%, 200 microl intra-articular injection) was diminished by 46.1 +/- 12.69% and 40.9 +/- 4.93% when the sciatic nerve was stimulated in the contralateral leg at 0.5 Hz for 1 h or 0.1 Hz for 4 h, respectively. 4. Stimulation of the peripheral stump of the left vagal nerve (20 V, 1 ms, 8 Hz, 10 min) elicited plasma extravasation in the trachea, oesophagus and mediastinal connective tissue in rats pretreated with atropine (2 mg kg(-1), i.v.), guanethidine (8 mg kg(-1), i.p.) and pipecuronium (200 microg kg(-1), i.v.). These responses were inhibited by 37.8 +/- 5.1%, 49.7 +/- 9.9% and 37.6 +/- 4.2%, respectively by antidromic sciatic nerve excitation (5 Hz, 5 min) applied 5 min earlier. 5. Pretreatment with polyclonal somatostatin antiserum (0.5 ml/rat, i.v.) or the selective somatostatin depleting agent cysteamine (280 mg kg(-1), s.c.) prevented the anti-inflammatory effect of sciatic nerve stimulation (5 Hz, 5 min) on a subsequent neurogenic plasma extravasation of the contralateral paw skin. The inhibitory effect of antidromic sciatic nerve excitation on plasma extravasation in response to vagal nerve stimulation was also prevented by somatostatin antiserum pretreatment. 6. Cutaneous blood flow assessment by laser Doppler flowmetry indicated that antidromic vasodilatation induced by sciatic nerve stimulation was not inhibited by excitation of the sciatic nerve of the contralateral leg (1 Hz, 30 min) or by somatostatin (10 microg/rat, i.v.) injection. 7. Plasma levels of somatostatin increased more than 4 fold after stimulation of both sciatic nerves (5 Hz, 5 min) but the stimulus-evoked increase was not observed in cysteamine (280 mg kg(-1), s.c.) pretreated rats. 8. These results suggest that somatostatin released from the activated sensory nerve terminals mediates the systemic anti-inflammatory effect evoked by stimulating the peripheral stump of the sciatic nerve.

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 microM) 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 submaximal 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 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg 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 pKB 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 Gi/Go proteins.

1. We have functionally characterized the human recombinant somatostatin (SRIF) sst5 receptor in Chinese hamster ovary-K1 (CHOsst5) cells by measuring total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 10 mM LiCl, in cells labelled with [3H]-myo-inositol. 2. In CHOsst5 cells, SRIF, SRIF-28 and the cyclic hexapeptide, L-362,855, produced time- and concentration-related increases in [3H]-InsPx accumulation, with similar potency (pEC50 values of 6.5, 6.8 and 7.2, respectively). L-362,855 behaved as a partial agonist, producing approximately 30% of the SRIF maximum response. The other peptide analogues of SRIF, BIM-23027 and BIM-23056, were inactive as agonists. 3. Increasing concentrations of L-362,855 increased [3H]-InsPx accumulation and simultaneously produced rightward shifts of SRIF concentration-effect curves, with an estimated pKp value of 7.6, confirming that it was acting as a partial agonist. 4. BIM-23056, but not BIM-23027, potently antagonized SRIF-induced [3H]-InsPx accumulation, with an estimated pKB value of 7.4. BIM-23056 did not antagonize [3H]-InsPx accumulation induced by uridine 5'-triphosphate (UTP). 5. SRIF- but not UTP-induced [3H]-InsPx accumulation was inhibited by increasing concentrations of pertussis toxin (0.01-100 ng ml-1), indicating the involvement of pertussis toxin-sensitive G-proteins. 6. These findings show that the human recombinant sst5 receptor, when stably expressed in CHO-K1 cells, is able to mediate activation of phosphoinositide metabolism in a pertussis toxin-sensitive manner. In this system L-362,855 behaved as a partial agonist while BIM-23056 was a specific antagonist. These agents should provide useful tools for functionally characterizing endogenous SRIF receptors.

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF1-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [125I]-Tyr11-SRIF-14, with a pIC50 of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF1 receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst2 subtype. 2. The stable radioligand, [125I]-BIM-23027, bound with high affinity (Kd = 13 pM, Bmax = 0.2 pmol mg-1 protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4 degrees C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15 degrees C and 89% of the ligand dissociated at 37 degrees C. 3. Equilibrium binding of [125I]-BIM-23027 was partially (25%) inhibited by 10 microM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [125I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 microM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4 degrees C, 81% at 15 degrees C and 93% at 37 degrees C. Binding was totally inhibited by pretreatment of cells with pertussis toxin. 4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC50 of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst2 receptor, no rise in intracellular calcium concentration was observed with SRIF-14. 5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst2 type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.

The transcription factors CREB (cAMP response element binding protein) and ATF (activating transcription factor) recognize DNA containing the consensus sequence TGACGTCA. We compared the neuropeptide somatostatin promoter, which binds CREB and is activated by cAMP, to the adenovirus E2A promoter, which binds ATF but is not activated by cAMP, to determine which specific nucleotides within a CREB/ATF recognition sequence confer cAMP responsiveness. Several mutant somatostatin promoters were generated containing part or all of the E2A ATF binding site. Some of the hybrid CREB/ATF binding sites competed for factor binding to a wild-type somatostatin promoter probe. However, only the wild-type CREB binding site promoter could confer cAMP activation on a linked CAT plasmid. Furthermore, this wild-type CREB binding site could confer cAMP activation on the CAT plasmid only if it was adjacent to a wild-type somatostatin TATA box and cap site. These results suggest that slight deviation from a wild-type CREB recognition sequence might be tolerated by factor(s) binding to cAMP response element-like sequences. However, transcription activation may require a particular CREB recognition sequence, as well as additional promoter elements that bind proteins that interact with CREB.