Abstract
Abstract: :
Purpose: The phototransduction cascade in the horseshoe crab Limulus polyphemus is not fully understood. Intracellular injection of cGMP or application of cGMP to excised patches activates a conductance similar to that activated by light, suggesting a role for cGMP. To further test this hypothesis, we have characterized inhibition of excitation resulting from the intracellular injection of inhibitors of guanylate cyclase. Methods: Photoreceptors were impaled with one or two microelectrodes to record membrane potential and inject drugs. Light or intracellular injection of 3'-deoxy-inositol-1,4,5-trisphosphate (3dIP3) was used to excite cells. Results: Competitive inhibitors of guanylate cyclase, such as guanosine-5'-tetraphosphate (GtetP), strongly and reversibly reduce the light response up to 95%. We investigated a second inhibitory mechanism using the P-site inhibitor 2'-deoxy-3'-guanosine monophosphate and phosphonoacetate. Either agent by itself had little or no effect on the light response; in combination they reduced the light response up to 90%, N = 9. To further test whether these inhibitors affect cyclase activity, we asked whether GtetP could block the depolarization induced by the phosphodiesterase inhibitor IBMX. On average, GtetP slowed the onset 60% and decreased the amplitude 55% of depolarization, N = 9. To test whether cyclase inhibitors act at an early or late stage of excitation, specifically before or after the light-dependent production of IP3; we asked whether GtetP blocks the excitation produced by 3dIP3. We found that GtetP decreased the excitation produced by 3dIP3 by an average of 70%, N = 5. Conclusions: Guanylate cyclase inhibitors acting by two mechanisms inhibited excitation consistent with a specific role for guanylate cyclase in phototransduction. GtetP counteracted the depolarization caused by IBMX, presumably by slowing and limiting a rise in cGMP levels. We conclude that a rise in cGMP levels is a late event in excitation, as GtetP strongly decreased the excitation produced by 3dIP3. One simple hypothesis is that IP3-mediated Ca2+ release leads to activation of guanylate cyclase resulting in accumulation of cGMP and opening of the light-dependent channels.
Keywords: 577 second messengers: pharmacology/physiology • 517 photoreceptors • 394 electrophysiology: non-clinical