Abstract
The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.
Highlights
The cGMP-dependent protein kinase (PKG)1 is the main mediator of nitric oxide (NO) induced relaxation of smooth muscle [1, 2]
The relaxant effect of cGMP in intact smooth muscle of the small intestine involves a decrease in intracellular [Ca2ϩ] as well as a reduced Ca2ϩ sensitivity of the contractile activation
We show using ␣-toxin-permeabilized preparations of cGKIϪ/Ϫ mice that the Ca2ϩ-desensitizing effect of 8-Br-cGMP in small intestinal smooth muscle is dependent on activation of PKG
Summary
The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide (NO) induced relaxation of smooth muscle [1, 2]. This pathway is well established, the cellular actions of PKG, NO, and cGMP are complex and not fully understood. Experiments on chemically skinned preparations where effects on Ca2ϩ sensitivity can be directly examined have revealed that cGMP lowers force at constant [Ca2ϩ] (14 –16). A detailed characterization of the cGMP and cAMP dependencies is difficult in intact tissue in which diffusion and metabolism of nucleotide analogues might influence the responses, and the effects on Ca2ϩ sensitivity cannot be directly assessed and distinguished from effects on intracellular Ca2ϩ levels. Using ␣-toxin-permeabilized preparations in which the concentration of free Ca2ϩ can be held constant, the effects of cGMP and cAMP on Ca2ϩ sensitivity were examined
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