Nitric oxide relaxes rat tail artery smooth muscle by cyclic GMP-independent decrease in calcium sensitivity of myofilaments

Abstract

The effects of authentic nitric oxide (NO, 10 −6 M) and NO-donors such as sodium nitroprusside (SNP, 10 −5 M) and glyceryl trinitrate (GTN, 10 −4 M) on contractile force and free intracellular calcium level ([Ca 2+] i) were studied on precontracted with high potassium chloride (KCl, 70 mM) isolated rings of rat tail artery. The sensitivity of contractile myofilaments to Ca 2+ was measured using chemically permeabilized (α-toxin, β-escin, Triton X-100) vascular rings. [Ca 2+] i and contractile activity were measured simultaneously. The relationship of [Ca 2+] i and tension developed was studied in endothelium-denuded rings and controlled calcium response was evaluated in both endothelium-denuded and permeabilized vascular rings. Both authentic NO and NO-donors decreased [Ca 2+] i and high potassium-induced tension with a different time course. Inhibitor of soluble guanylyl cyclase (sGC) LY83583 (10 −5 M) did not affect SNP-induced relaxation whereas the other sGC inhibitor ODQ (10 −6 M) attenuated SNP-induced relaxation. Both inhibitors had no effect on NO- and SNP-induced reduction in [Ca 2+] i. On the contrary, GTN induced neither relaxation nor decrease in [Ca 2+] i on application of both LY83583 and ODQ. Tail artery rings permeabilized with α-toxin, β-escin, but not with Triton X-100 were relaxed by authentic NO and NO-donors, but to a less extent than non-permeabilized rings. Dithioerythritol (DTE, 5×10 −3 M) that maintains sulfhydryl (SH) groups in reduced state preventing their nitrosylation attenuated NO-induced relaxation in both non-permeabilized and permeabilized tail artery rings. The cyclic heptapeptide mycrocystin-LR (MC-LR) (10 −5 M), an inhibitor of type 1 and 2A phosphatases, induced sustained increase in tension of β-escin permeabilized rings in low Ca 2+ (10 −8 M) solution. The tension was not affected by authentic NO and SNP. We conclude that authentic NO and SNP relax rat tail artery smooth muscle (SM) in the presence of inhibitors of sGC via cyclic guanosine monophosphate (cGMP)-independent pathway, whereas relaxation induced by GTN is inhibited. The data demonstrate that cGMP-dependent pathway in vascular smooth muscle is ubiquitous, but not the only way of relaxation induced by NO. NO can modulate vascular tone directly by reducing sensitivity of contractile myofilaments to [Ca 2+] i and may involve activation of protein phosphatase(s).