Forskolin upregulation of NOS I protein expression in porcine ciliary processes: a new aspect of aqueous humor regulation

Zusammenfassung

Hintergrund: Ziel der vorliegenden Arbeit war es, den Einfluss von Forskolin (Adenylatzyklasestimulator, 1 µM) auf die Expression der Stickstoffmonoxidsynthase (NOS) auf Proteinebene zu untersuchen. Methode: Die frisch exzidierten Schweineziliarkörper wurden homogenisiert. Mittels monoklonalen Antikörpern wurden im Western-Blot neuronale NOS (nNOS, NOS I), endotheliale NOS (ecNOS, NOS III) sowie makrophage NOS (macNOS, NOS II) identifiziert. Zusätzlich wurden die Gewebe für 4 - 24 Stunden mit Forskolin (1 µM) inkubiert und wiederum die Expression von neuronaler NOS und makrophager NOS untersucht. Ergebnisse: Das neuronale NOS-Protein wird im Schweineziliarkörper stark exprimiert. Im Vergleich zur neuronalen NOS, zeigten endotheliale NOS und makrophage NOS eine weniger starke Expression. Im Vergleich zu den Kontrollwerten zeigte sich unter Forskolin eine erhöhte Menge des neuronalen NOS-Proteins. Schlussfolgerungen: Im Schweineziliarkörper sind drei Stickstoffmonoxidsynthasen vorhanden. Das neuronale NOS-Isoenzym zeigt die stärkste Expression, welche durch Zugabe von Forskolin noch zusätzlich gesteigert werden kann.

Abstract

Background: To determine whether nitric oxide synthase (NOS) protein expression can be modulated by forskolin in porcine ciliary processes. Method: Western blot analysis using mouse monoclonal antibodies against NOS I (neuronal NOS or nNOS), NOS II (macrophage NOS or macNOS), and NOS III (endothelial NOS or eNOS) was performed in porcine ciliary processes. Expression of NOS I and II was also assessed in the presence or in the absence of 1 µM forskolin exposure (4 and 24 hours). Results: All three NOS isoforms could be detected in isolated porcine ciliary processes. Protein expression for NOS I was about 6 times higher than for NOS III and 15 times higher than for NOS II. In comparison to controls, after forskolin exposure, NOS I protein expression was increased by about 1.5-fold. No change could be observed in NOS II protein expression after forskolin exposure. Conclusions: Nitric oxide is involved in transepithelial fluid transport of different organs (lung, kidney, colon, etc.) and is suspected to play a role in ciliary processes by modulating the aqueous humor production. The present study indicates that forskolin (which increases cAMP production) modulates NOS I protein expression in isolated porcine ciliary processes. These results suggest that selective inhibition of NOS I could be tried for reducing aqueous humor production. The clinical relevance requires further investigations.

Schlüsselwörter

Glaukom - Stickstoffmonoxid - Kammerwasser - Augeninnendruck

Key words

Glaucoma - intraocular pressure - nitric oxide - aqueous humor production

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