Effect of somatostatin on nitric oxide production in human retinal pigment epithelium cell cultures.
OBJECTIVE
To investigate the presence of somatostatin and its receptors (sst(1-5) receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures.


METHODS
Human RPE cells (D407) were used for all studies performed. Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5). Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for [125I]Tyr11 somatostatin binding. The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2- and NO3- (NOx-).


RESULTS
SRIF was detected in a concentration of 0.56 +/- 0.13 picomoles/mg protein. sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures. sst1- and sst5- but not sst2-selective ligands displaced the specific [125I]Tyr11 somatostatin binding to RPE cell membranes. NADPH-diaphorase stain and iNOS mRNA were detected. SRIF and the sst2-selective analogue MK678 increased the levels of NOx- in a concentration-dependent manner. This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2).


CONCLUSIONS
These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin. Somatostatin's ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.
Vasilaki
Anna
A
Department of Pharmacology, Faculty of Medicine, University of Crete, Heraclion, Crete, Greece.
Papadaki
Thekla
T
Notas
George
G
Kolios
George
G
Mastrodimou
Niki
N
Hoyer
Daniel
D
Tsilimbaris
Miltiadis
M
Kouroumalis
Elias
E
Pallikaris
Ioannis
I
Thermos
Kyriaki
K
D001665:Binding Sites; D018929:Cell Culture Techniques; D004305:Dose-Response Relationship, Drug; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011863:Radioimmunoassay; D011869:Radioligand Assay; D017481:Receptors, Somatostatin / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*
D006727:Hormone Antagonists; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D009569:Nitric Oxide; D013004:Somatostatin; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D009252:NADPH Dehydrogenase