Davide Cervia

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

Somatostatin (SRIF)‐14 is recognized as an important mediator between the nervous and the immune system, although the functional role of its receptors (sst1–sst5) is poorly understood in humans. In our study, we demonstrate that human macrophages, differentiated from PBMC‐derived monocytes, express sst1 and sst2 mRNAs. sst1 and sst2 are mostly localized at the cell surface and display active binding sites. In particular, sst1/sst2 activation results in a weak internalization of sst1, and the sst2 internalization appears more efficient. At the functional level, the activation of SRIF receptors by the multiligand analogs SOM230 and KE108, but not by SRIF‐14 or cortistatin‐14, reduces macrophage viability. Their effects are mimicked by the selective activation of sst1 and sst2 using CH‐275 and SMS 201‐995/L‐779,976, respectively. Further, sst1‐ and sst2‐mediated effects are reversed by the sst1 antagonist SRA‐880 or the sst2 antagonist CYN 154806, respectively. CH‐275, SMS 201‐995, and L‐779,976, but not SRIF‐14, decrease mRNA expression and secretion of the MCP‐1. In addition, SRIF‐14, CH‐275, SMS 201‐995, and L‐779,976 decrease IL‐8 secretion, and they do not affect IL‐8 mRNA expression. In contrast, SRIF‐14 and sst1/sst2 agonists do not affect the secretion of matrix metalloproteinase‐9. Collectively, our results suggest that the SRIF system, through sst1 and sst2, exerts mainly an immunosuppressive effect in human macrophages and may, therefore, represent a therapeutic window that can be exploited for the development of new strategies in pharmacological therapy of inflammation.

Genetic deletion of somatostatin receptor 1 alters somatostatinergic transmission in the mouse retina

In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst(1-5)). Among them, the sst1 receptor has been localised to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localisation and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localised to SRIF-containing amacrine cells, whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF, whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain.

Physiology and pathology of somatostatin in the mammalian retina: a current view.

In the retina, peptidergic signalling participates in multiple circuits of visual information processing. The neuropeptide somatostatin (SRIF) is localised to amacrine cells and, in some instances, in a subset of ganglion cells. The variegated expression patterns of SRIF receptors (sst(1)-sst(5)) and the variety of signalling mechanisms activated by retinal SRIF suggest that this peptide may exert multiple actions on retinal neurons and on retinal physiology, although our current understanding reflects a rather complicated picture. SRIF, mostly through sst(2), may act as a positive factor in the retina by regulating retinal homeostasis and protecting neurons against damage. In this respect, SRIF analogues seem to constitute a promising therapeutic arsenal to cure different retinal diseases, as for instance, ischemic and diabetic retinopathies. However, further investigations are needed not only to fully understand the functional role of the SRIF system in the retina but also to exploit new chemical space for drug-like molecules.

Somatostatin-induced control of cytosolic free calcium in pituitary tumour cells

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. 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. Pretreatment with octreotide or with L‐Tyr8Cyanamid 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. sst1 and sst2 receptor‐immunoreactivities (‐IRs) are localized to cell membranes. sst2, but not sst1 receptor‐IR, internalizes after cell exposure to octreotide. SRIF‐induced inhibition of basal [Ca2+]i or FSK‐induced Ca2+ entry is blocked by pertussis toxin (PTX). 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. 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. 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.

Pharmacological characterisation of native somatostatin receptors in AtT-20 mouse tumour corticotrophs

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 (sst1 – sst5). 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. [125I]LTT‐SRIF‐28, [125I]CGP 23996, [125I]Tyr10‐cortistatin‐14 and [125I]Tyr3‐octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (Bmax=315, 274, 239 and 206 fmol mg−1, respectively). [125I]LTT‐SRIF‐28 labels significantly more sites than [125I]Tyr10 ‐cortistatin‐14 and [125I]Tyr3 ‐octreotide as seen previously in cells expressing pure populations of sst2 or sst5 receptors. SRIF analogues displaced the binding of the four radioligands. sst2/5 receptor‐selective ligands showed much higher affinity than sst1/3/4 receptor‐selective ligands. The binding profile of [125I]Tyr3‐octreotide was different from that of [125I]LTT‐SRIF‐28, [125I]CGP 23996 and [125I]Tyr10‐cortistatin‐14. The sst5/1 receptor‐selective ligand L‐817,818 identified two binding sites, one with subnanomolar affinity (sst5 receptors) and one with micromolar affinity (sst2 receptors); however, the proportions were different: 70 – 80% high affinity with [125I]LTT‐SRIF‐28, [125I]CGP 23996, [125I]Tyr10‐cortistatin‐14, but only 20% with [125I]Tyr3‐octreotide. SRIF analogues inhibited the forskolin‐stimulated cAMP levels depending on concentration. sst2/5 receptor‐selective ligands were highly potent, whereas sst1/3/4 receptor‐selective ligands had no significant effects. The sst2 receptor antagonist D‐Tyr8‐CYN 154806 competitively antagonised the effects of SRIF‐14 and sst2 receptor‐preferring agonists, but not those of L‐817,818. The complex binding properties of SRIF receptor analogues indicate that sst2 and sst5 receptors are the predominant SRIF receptors expressed on AtT‐20 cell membranes with no or only negligible presence of sst1, sst3 and sst4 receptors. In the functional studies using cAMP accumulation, only sst2 and sst5 receptors appear to play a role. However, the ‘predominant’ receptor appears to be the sst2 receptor, although sst5 receptors can also mediate the effect, when the ligand is not able to activate sst2 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.

Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices

Somatostatin‐14 [somatotropin release‐inhibiting factor (SRIF)] reduces hippocampal epileptiform activity but the contribution of its specific receptors (sst1−5) is poorly understood. We have focused on the role of sst1 and sst2 in mediating SRIF modulation of epilepsy using hippocampal slices of wild‐type (WT) and sst1 or sst2 knockout (KO) mice. Recordings of epileptiform discharge induced by Mg2+‐free medium with 4‐aminopyridine were performed from the CA3 region before and after the application of SRIF compounds. In WT mice, SRIF and the sst1 agonist CH‐275 reduce epilepsy whereas sst1 blockade with its antagonist SRA‐880 increases the bursting discharge. Activation of sst2 does not affect the bursting frequency unless its agonist octreotide is applied with SRA‐880, indicating that sst1 masks sst2‐mediated modulation of epilepsy. In sst1 KO mice: (i) the bursting frequency is lower than in WT; (ii) SRIF, CH‐275 and SRA‐880 are ineffective on epilepsy and (iii) octreotide is also devoid of effects, whereas blockade of sst2 with the antagonist d‐Tyr8 Cyn 154806 increases the bursting frequency. In sst2 KO mice, the SRIF ligand effects are similar to those in WT. In the whole hippocampus of sst1 KO mice, sst2 mRNA, protein and binding are higher than in WT and reverse transcription‐polymerase chain reaction of the CA3 subarea confirms an increase of the sst2 messenger. We conclude that sst1 mediates inhibitory actions of SRIF and that interactions between sst1 and sst2 may prevent sst2 modulation of epilepsy. We suggest that, in sst1 KO mice, activation of over‐expressed sst2 reduces the bursting frequency, indicating that sst2 density represents the rate‐limiting factor for sst2‐mediated modulation of epilepsy.

Modulation of the neuronal response to ischaemia by somatostatin analogues in wild‐type and knock‐out mouse retinas

Somatostatin acts at five G protein‐coupled receptors, sst1‐sst5. In mouse ischaemic retinas, the over‐expression of sst2 (as in sst1 knock‐out mice) results in the reduction of cell death and glutamate release. In this study, we reported that, in wild‐type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischaemia‐induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over‐expressing ischaemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti‐ischaemic effect of somatostatin agonists in the presence of sst2 over‐expression, we tested sst2 desensitisation because of internalisation or altered receptor function. We observed that (i) sst2 was not internalised, (ii) among G protein‐coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischaemia, (iii) both GRK1 and RGS1 were down‐regulated by octreotide in wild‐type ischaemic retinas, (iv) octreotide down‐regulated GRK1 but not RGS1 in sst2 over‐expressing ischaemic retinas. These results demonstrate that sst2 activation protects against retinal ischaemia. However, in the presence of sst2 over‐expression sst2 is functionally desensitised by agonists, possibly because of sustained RGS1 levels.

Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission

Somatostatin‐14 (SRIF) co‐localizes with γ‐aminobutyric acid (GABA) in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of seizures has been proposed, although its exact contribution requires some clarification. In particular, SRIF knockout (KO) mice do not exhibit spontaneous seizures, indicating that compensatory changes may occur in KO. In the KO hippocampus, we examined whether specific SRIF receptors and/or the cognate peptide cortistatin‐14 (CST) compensate for the absence of SRIF. We found increased levels of both sst2 receptors (sst2) and CST, and we explored the functional consequences of sst2 compensation on bursting activity and synaptic responses in hippocampal slices. Bursting was decreased by SRIF in wild‐type (WT) mice, but it was not affected by either CST or sst2 agonist and antagonist. sst4 agonist increased bursting frequency in either WT or KO. In WT, but not in KO, its effects were blocked by agonizing or antagonizing sst2, suggesting that sst2 and sst4 are functionally coupled in the WT hippocampus. Bursting was reduced in KO as compared with WT and was increased upon application of sst2 antagonist, while SRIF, CST and sst2 agonist had no effect. At the synaptic level, we observed that in WT, SRIF decreased excitatory postsynaptic potentials which were, in contrast, increased by sst2 antagonist in KO. We conclude that sst2 compensates for SRIF absence and that its upregulation is responsible for reduced bursting and decreased excitatory transmission in KO mice. We suggest that a critical density of sst2 is needed to control hippocampal activity.

Vascular endothelial growth factor in the ischemic retina and its regulation by somatostatin

J. Neurochem. (2012) 120, 818–829.

Inhibitory Control of Growth Hormone Secretion by Somatostatin in Rat Pituitary GC Cells: sst2 but Not sst1 Receptors Are Coupled to Inhibition of Single-Cell Intracellular Free Calcium Concentrations

Rat pituitary tumor cells (GC cells) exhibit spontaneous oscillations of intracellular free calcium concentration ([Ca²⁺]_i) that allow continuous release of growth hormone (GH). Of the somatostatin (SRIH) receptor subtypes (sst receptors) mediating SRIH action, sst₁ and sst₂ receptors are highly expressed by GC cell membranes. In the present study, the effects of sst₁ or sst₂ receptor activation on single-cell [Ca²⁺]_i were investigated in GC cells by confocal fluorescence microscopy. In addition, the effects of sst₁ or sst₂ receptor activation on GH secretion were also studied. Our results demonstrate that SRIH decreases [Ca²⁺]_i baseline and almost completely blocks Ca²⁺ transients through activation of sst₂ but not of sst₁ receptors. In contrast, SRIH effectively inhibits GH secretion through activation of both sst₁ and sst₂ receptors. Blocking Ca²⁺ transients is less efficient than SRIH to inhibit GH release. The cyclic octapeptide, CYN-154806, antagonizes sst₂ receptors at [Ca²⁺]_i since it abolishes the sst₂ receptor-mediated inhibition of [Ca²⁺]_i without affecting single-cell Ca²⁺ signals. On the other hand, CYN-154806 alone potently inhibits GH secretion through the involvement of pertussis toxin-sensitive G proteins. In conclusion, the present results demonstrate that SRIH inhibition of GH release in GC cells involves mechanisms either dependent or independent on SRIH modulation of [Ca²⁺]_i. The implications of CYN-154806 inhibition of GH secretion are discussed.