Nitric oxide synthase activity in retinas from non-insulin-dependent diabetic Goto-Kakizaki rats: correlation with blood-retinal barrier permeability.
The aim of this work was to examine whether the non-insulin-dependent diabetic Goto-Kakizaki (GK) rats develop retinal changes with similar characteristics to those observed in insulin-dependent diabetic rats in what concerns blood-retinal barrier (BRB) permeability, nitric oxide (NO) production, and retinal IL-1beta level. BRB permeability was evaluated by vitreous fluorophotometry. NO synthase (NOS) activity was assessed by the production of l-[(3)H]-citrulline and retinal IL-1beta level was determined by ELISA. The expression of the inducible isoform of NOS (iNOS) protein was evaluated by Western blot analysis and immunohistochemistry. The in vivo studies indicated that in GK rats the BRB permeability to fluorescein was increased (787.81 +/- 68 min(-1)) in comparison to that in normal Wistar rats (646.6 +/- 55 min(-1)). The ex vivo studies showed that in retinas from GK rats the NOS activity was higher (207 +/- 28.9 pmol l-[(3)H]-citrulline/mg protein/30 min) than that in normal Wistar rats (125 +/- 32.3 pmol l-[(3)H]-citrulline/mg protein/30 min). These results were correlated with an increase in the protein level of iNOS in the retinas of GK rats, which was confirmed not only by the study of the iNOS protein expression but also by the use of NOS activity inhibitors. Indeed, the data about the effect of specific inhibitors on the NOS activity revealed that in retinas from GK rats the most effective inhibitor was aminoguanidine, which predominantly inhibits the iNOS isoform whereas in retinas from normal Wistar rats it was N(G) nitro l-arginine that predominantly inhibits the constitutive isoforms of NOS. In summary, in retinas from GK rats there is an increased production of NO which may contribute to the BRB breakdown.
Carmo
A
A
Center of Opthalmology, Institute for Biomedical Research on Light and Image, University of Coimbra, Coimbra, Portugal. analia@imagem.ibili.uc.pt
Cunha-Vaz
J G
JG
Carvalho
A P
AP
Lopes
M C
MC
D000818:Animals; D001120:Arginine / Q000378:metabolism / Q000493:pharmacokinetics; D001786:Blood Glucose; D001813:Blood-Retinal Barrier / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D003720:Densitometry; D003921:Diabetes Mellitus, Experimental / Q000150:complications / Q000201:enzymology*; D003924:Diabetes Mellitus, Type 2 / Q000150:complications / Q000201:enzymology*; D003930:Diabetic Retinopathy / Q000201:enzymology* / Q000209:etiology / Q000473:pathology; D004195:Disease Models, Animal; D005451:Fluorescein Angiography; D006146:Guanidines / Q000494:pharmacology; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D019335:Nitroarginine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D017208:Rats, Wistar; D012160:Retina / Q000201:enzymology / Q000473:pathology; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D001786:Blood Glucose; D006146:Guanidines; D007375:Interleukin-1; D019335:Nitroarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; C004479:pimagedine