Role of G-proteins in ciliary process adenylyl cyclase responses of the albino rabbit eye

Abstract

After intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5–1 μg/eye) into the albino rabbit eye, the in vitro responses of ciliary process adenylyl cyclase (AC) to isoproterenol, vasoactive intestinal peptide (VIP), and forskolin (FSK) were increased 21–40% for PTX, but for CTX-injected eyes AC responses to fluoroaluminate, VIP and FSK decreased 70–50%. The increased responses after PTX suggests that this toxin blocked an inhibitory G_i control of AC that is present in the control tissue. However, prolonged (> 24 hr) in vivo exposure to CTX appears to down-regulate the AC enzyme. In contrast to the in vivo findings, AC responsiveness was unaffected by PTX pre-treatment of membranes in vitro, while CTX pre-treatment increased basal activity (+600%), and the FSK response (+30%), but decreased responsiveness to fluoroaluminate, VIP and isoproterenol by 88–56%.

Treatment of ciliary process membranes with ³²P-NAD and CTX or PTX followed by SDS-PAGE autoradiography of labelled proteins gave two bands for the G-protein α-subunits of Gs (45,56 kDa) and one broad band centered at 40 kDa for G_i-type subunits respectively. Western blots using specific antibodies showed the presence of G_i type I or III, but no detectable G_i type II or G_o in rabbit ciliary processes. We conclude that the changes in adenylyl cyclase enzyme responses after intraocular CTX or PTX may not correlate with cAMP levels and intraocular pressure effects. However, the in vitro biochemical data on AC responses and on G-proteins provide evidence for dual regulation of ciliary process AC by activating and inhibitory G-proteins.

Key Words:

• G-proteins
• adenylyl cyclase
• ciliary process
• intraocular pressure
• cholera toxin
• pertussis toxin