Pharmacology of the nitric oxide receptor, soluble guanylyl cyclase, in cerebellar cells

Abstract

The nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), is commonly manipulated pharmacologically in two ways. Inhibition of activity is achieved using 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) which oxidizes the haem prosthetic group to which NO binds, while the compound 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1) is considered an 'allosteric' activator. Knowledge of how these agents function and interact in a normal cellular environment is limited. These issues were addressed using rat cerebellar cells. Inhibition by ODQ was not simply competitive with NO. The rate of onset was ODQ concentration-dependent and developed in two kinetic phases. Recovery from inhibition occurred with a half-time of approximately 5 min. YC-1 slowed the rate at which sGC deactivated on removal of NO by 45 fold, consistent with YC-1 increasing the potency of NO for sGC. YC-1 also enhanced the maximal response to NO by 2 fold. Furthermore, when added to cells in which sGC was 90% desensitized, YC-1 abruptly enhanced sGC activity to a degree that indicated partial reversal of desensitization. After pre-exposure to YC-1, sGC became resistant to inhibition by ODQ. In addition, YC-1 rapidly reversed inhibition by ODQ in cells and for purified sGC, suggesting that YC-1 either increases the NO affinity of the oxidized sGC haem or reverses haem oxidation. It is concluded that the actions of ODQ and YC-1 on sGC are broadly similar in cells and purified preparations. Additionally, YC-1 transiently reverses sGC desensitization in cells. It is hypothesized that YC-1 has multiple actions on sGC, and thereby both modifies the NO binding site and enhances agonist efficacy.

Figure 1

Inhibition of sGC by ODQ in cerebellar cells. (a) Concentration-response curve for ODQ in cells exposed to 1 μM DEA/NO for 2 min. Cells were preincubated with ODQ for 10 min. (b) Concentration-response curve for DEA/NO following 20 min preincubation in the absence (control) and presence of 0.3 μM ODQ. (c) Time courses for onset of inhibition. Cells were preincubated with ODQ at the concentrations and for the times illustrated, before exposure to 1 μM DEA/NO for 2 min. (d) Recovery of cells from ODQ inhibition. Cells were preincubated for 10 min with 0.3 μM ODQ, then diluted 1 in 10 into buffer lacking ODQ. After various times the cells were exposed to 1 μM DEA/NO for 2 min. Recovery was assessed by comparison with the cyclic GMP response in cells treated with 0.3 μM and 0.03 μM ODQ when diluted into buffer containing an equal concentration of ODQ (columns).

Figure 2

Effects of YC-1 on sGC. YC-1 was applied at various concentrations for 10 min to control (untreated) cells, cells preincubated (10 min) with 100 μM L-NA, and cells preincubated (10 min) with 10 μM Hb, as indicated. Also shown are the cyclic GMP responses of cells that were preincubated with 100 μM L-NA (10 min) and YC-1 (5 s) and then exposed to 1 μM DEA/NO for 2 min. Column indicates response to 1 μM DEA/NO alone.

Figure 3

Effect of YC-1 on the rate of deactivation of sGC. (a) Cells were exposed to 1 μM DEA/NO at t=0, in the absence (control) or presence of 100 μM YC-1 (5 s preincubation) as indicated. After 5 s (arrow) 10 μM Hb was added to effect deactivation of sGC. The decline in cyclic GMP accumulation in YC-1-treated cells is fitted with an exponential. (b) Addition of Hb or ODQ (arrow) to control and YC-1-treated cells after 2 min DEA/NO exposure. The continuous line is a fit to control data with the integrated Michaelis-Menten equation. Broken line is the predicted decline in cyclic GMP in YC-1 treated cells given the Michaelis-Menten parameters determined for control cells.

Figure 4

Effect of YC-1 at steady-state cyclic GMP levels. Steady-state cyclic GMP levels were achieved in untreated (control), YC-1 pre-treated (100 μM, 5 s), and ODQ pre-treated (10 μM, 5 s) cells by addition of DEA/NO (ascending arrows) at 0 and 2 min. Inset shows the corresponding NO concentration profile. At steady-state, 100 μM YC-1 was added (at descending arrows) to control and ODQ-treated cells. Dashed line indicates a 2 fold enhancement of the 10% of sGC that is non-desensitized at steady-state, assuming zero PDE activity and no further desensitization (i.e. the maximum possible rate of cyclic GMP synthesis predicted for a simple mechanism of YC-1 action; see Discussion).

Figure 5

Interaction between YC-1 and ODQ on purified sGC. (a) Time course of the activity of purified sGC (50 ng ml⁻¹) exposed to 100 μM DEA/NO in the absence (control) and presence of 100 μM YC-1, and following addition (arrow) of ODQ (1 μM) in the presence of YC-1. (b) Time course of sGC activity in the presence of 1 μM ODQ with or without addition of 100 μM YC-1 (arrow). The broken line represents the slope predicted if the degree of potentiation was the same as that found for uninhibited sGC. Numbers in brackets are rates in μmol cyclic GMP.mg sGC⁻¹.min⁻¹.

Figure 6

Model for the interaction of the NO receptor, sGC, with agonist (NO) and pharmacological modulators (ODQ and YC-1). The NO receptor exists in equilibrium between free (NO+sGC), bound (NO-sGC), active (NO-sGC^*), and desensitized states (NO-sGC^d), and also between ferrous (sGC_II) and ferric (sGC_III) states. The inhibitor ODQ oxidizes ferrous sGC to the ferric form. YC-1 has multiple effects on sGC function, illustrated for simplicity as modulation of the transitional steps between states (as numbered; see Discussion for details). Dashed lines illustrate hypothesized routes to the desensitized form.