cyn-154806 and Disease-Models--Animal

Somatostatin (SST) and some of its receptor subtypes have been implicated in pain signaling at the spinal level. In this study we have investigated the role of SST and its sst2A receptor (sst2A) in dorsal root ganglia (DRGs) and spinal cord.. SST and sst2A protein and sst2 transcript were found in both mouse and human DRGs, sst2A-immunoreactive (IR) cell bodies and processes in lamina II in mouse and human spinal dorsal horn, and sst2A-IR nerve terminals in mouse skin. The receptor protein was associated with the cell membrane. Following peripheral nerve injury sst2A-like immunoreactivity (LI) was decreased, and SST-LI increased in DRGs. sst2A-LI accumulated on the proximal and, more strongly, on the distal side of a sciatic nerve ligation. Fluorescence-labeled SST administered to a hind paw was internalized and retrogradely transported, indicating that a SST-sst2A complex may represent a retrograde signal. Internalization of sst2A was seen in DRG neurons after systemic treatment with the sst2 agonist octreotide (Oct), and in dorsal horn and DRG neurons after intrathecal administration. Some DRG neurons co-expressed sst2A and the neuropeptide Y Y1 receptor on the cell membrane, and systemic Oct caused co-internalization, hypothetically a sign of receptor heterodimerization. Oct treatment attenuated the reduction of pain threshold in a neuropathic pain model, in parallel suppressing the activation of p38 MAPK in the DRGs. The findings highlight a significant and complex role of the SST system in pain signaling. The fact that the sst2A system is found also in human DRGs and spinal cord, suggests that sst2A may represent a potential pharmacologic target for treatment of neuropathic pain.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Glutamate Decarboxylase; Green Fluorescent Proteins; Humans; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Octreotide; Oligopeptides; Pain Threshold; Protein Transport; Receptors, Somatostatin; Sciatica; Sensory Receptor Cells; Somatostatin

The neuropeptide somatostatin and selective analogs for the sst(2/5) receptor subtypes provided neuroprotection against retinal chemical ischemia ex vivo and AMPA [(RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide] induced retinal toxicity in vivo, when employed in micromolar concentrations (Mastrodimou et al., 2005; Kiagiadaki and Thermos, 2008). The aim of the present study was to investigate the neuroprotective properties of a new metabolically stable agent pasireotide (SOM230) in the above mentioned retinal models of ischemia. Adult Sprague Dawley (250-350 g) rats were employed. For the ex vivo experiments, retinal eye cups were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanide (25 mM)] in the absence or presence of SOM230 (10(-7)-10(-5) M) alone or in the presence of the sst(2) antagonist CYN-154806 (10(-7) or 10(-5) M). In the in vivo model, the animals received intravitreally: PBS (50 mM), AMPA (42 nmol/eye) or AMPA (42 nmol) in combination with SOM230 (10(-7)-10(-5) M). Immunohistochemistry studies using antisera against bNOS, a marker for brain/neuronal NOS containing amacrine cells, protein kinase C (PKC) a marker for rod bipolar cells, and TUNEL studies in conjunction with FACS analysis were employed to examine retinal cell loss and protection. Chemical ischemia led to a loss of bNOS and PKC immunoreactivity which was reversed by SOM230. Partial and full protection of bNOS and PKC immunoreactive neurons, respectively, was observed even at the low concentration of 10(-7) M. The neuroprotective actions of SOM230 (10(-7) or 10(-5) M) were reversed by CYN-154806 (10(-7) or 10(-5) M, respectively). Similarly, SOM230 (10(-7), 10(-6), 10(-5) M) provided neuroprotection in the in vivo model. The dose of 10(-7) M prevented the loss of the bNOS cells and provided almost full protection. These data were substantiated by TUNEL staining and fluorescence-activated cell sorting (FACS) analysis. SOM230 appears very efficacious in its neuroprotective properties in both models of retinal ischemia affording neuroprotection at the concentration or dose of 100 nM. These data suggest that SOM230 might represent a useful pharmacological compound for the treatment of retinal disease.

Topics: Amacrine Cells; Animals; Apoptosis; Disease Models, Animal; Flow Cytometry; Fluorescent Antibody Technique, Indirect; In Situ Nick-End Labeling; Neuroprotective Agents; Nitric Oxide Synthase Type I; Oligopeptides; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Somatostatin; Reperfusion Injury; Retinal Diseases; Somatostatin

In a recent study, we employed an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatinergic agents. Intravitreal administration of somatostatin and sst(2) selective agonists protected the retina from (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) induced excitotoxicity. The sst(1) and sst(4) selective ligands had no effect (Kiagiadaki and Thermos, 2008). The presence of sst(5) receptors in rat retina was only recently reported (Ke and Zhong, 2007). Synthetic agonists that activate sst(2) receptors also bind with high affinity to the sst(5) subtype. In the present study the putative neuroprotective effects of sst(5) receptor activation were investigated. Adult female and male Sprague-Dawley (250-350g) rats were employed. Groups of animals received intravitreally PBS (50mM) or AMPA (42 nmol/eye) alone or in combination with L-817,818 (sst(5), 10(-5), 10(-4)M). To exclude neuroprotective effects via the activation of sst(2) receptors, L-817,818 (10(-4)M) was coinjected with the sst(2) antagonist CYN-154806 (10(-4)M). Immunohistochemistry (IHC) studies using the anti-retinal marker choline acetyltransferase (ChAT) and TUNEL staining were employed to examine retinal cell loss and protection. IHC and Western blot analysis were also employed to assess whether the sst(5) receptors are viable in the AMPA treated tissue as compared to control retina. sst(5) receptors were not affected by AMPA. L-817,818 protected the retina from the AMPA insult in the dose of 10(-4)M, while CYN-154806 (10(-4)M) had no effect on the sst(5) neuroprotection. TUNEL staining confirmed the AMPA-induced retinal toxicity and the L-817,818 neuroprotection. These results demonstrate for the first time that sst(5) receptors are functional in the retina, and that sst(5) analogs administered intravitreally protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amides; Animals; Cell Death; Choline O-Acetyltransferase; Disease Models, Animal; Excitatory Amino Acid Agonists; Female; Gene Expression Regulation; In Situ Nick-End Labeling; Male; Naphthalenes; Neuroprotective Agents; Neurotoxicity Syndromes; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptors, Somatostatin; Retina

Purpose. In the mouse model of oxygen-induced retinopathy (OIR), somatostatin-14 (SRIF) acting at the SRIF receptor subtype 2 (sst(2)) inhibits angiogenic responses to hypoxia through a downregulation of vascular endothelial growth factor. Information about where SRIF-sst(2) interactions take place is lacking, and downstream effectors mediating SRIF-sst(2) antiangiogenic actions are unknown. Methods. In the OIR model, retinal expression of SRIF was evaluated with RT-PCR and radioimmunoassay. The bindings of [(125)I]LTT-SRIF-28 and [(125)I]Tyr(3)-octreotide were measured in coronal sections of the eye. With Western blot analysis, the authors evaluated the levels of sst(2A) and the expression and activity of the signal transducer and activator of transcription (STAT)3. The analysis of STAT3 was performed in hypoxic mice treated with the sst(2) agonist octreotide or with the sst(2) antagonist D-Tyr(8) cyanamid 154806 (CYN). Retinal localization of sst(2A) was assessed by single and double immunohistochemistry with an endothelial cell marker. Results. In the hypoxic retina, both SRIF and sst(2) levels as well as [(125)I]Tyr(3)-octreotide binding were downregulated. In addition, sst(2A) immunostaining was decreased in the neuroretina but was increased in capillaries. Hypoxia increased both the expression and the activity of STAT3. This increase was inhibited by octreotide but was strengthened by CYN. Conclusions. These data suggest that sst(2) expressed by capillaries may be responsible for the antiangiogenic effects of SRIF and that downstream effectors in this action include the transcription factor STAT3. These results support the possibility of using sst(2)-selective ligands in the treatment of proliferative retinopathies and indicate STAT3 as an additional target for a novel therapeutic approach.

Topics: Animals; Animals, Newborn; Autoradiography; Blotting, Western; Disease Models, Animal; Female; Fluorescent Antibody Technique, Indirect; Humans; Hypoxia; Infant, Newborn; Male; Mice; Mice, Inbred C57BL; Octreotide; Oligopeptides; Oxygen; Radioimmunoassay; Receptors, Somatostatin; Retinal Vessels; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; Somatostatin; STAT3 Transcription Factor