Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway

Abstract

1. The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 microM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM). 3. In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. 4. In beta-escin skinned preparations, NO had no effect on tone induced by calcium (1 microM in the presence of 100 microM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.

Figure 1

Concentration-response curves for relaxations of the mouse anococcygeus muscle to (a) sodium nitroprusside (SNP) or (b) nitric oxide (NO), and (c) frequency-response curve for relaxations to nitrergic field stimulation, when tone was raised with either 50 μM carbachol or 100 nM thapsigargin. Each point is the mean±s.e.mean from a minimum of five individual muscle preparations. ^*P<0.05 compared with corresponding value with carbachol tone.

Figure 2

Concentration-response curves for relaxations of the mouse anococcygeus muscle to (a) sodium nitroprusside (SNP) and nitric oxide (NO) or (b) 8-Br-cyclic GMP and the effect on these of the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo [4,3-a]quinoxalin-1-one (ODQ; 5 μM). Tone was raised with 100 nM thapsigargin (Tg). Each point is the mean±s.e.mean from a minimum of five individual muscle preparations.

Figure 3

Recordings of the effects of carbachol (50 μM), thapsigargin (Tg; 100 nM) and sodium nitroprusside (SNP; 10 μM) on cytoplasmic calcium levels (measured as the Fura-2 ratio; R_340/380) in single smooth muscle cells of the mouse anococcygeus. Each trace comprises the integrated data from (a) 6 cells, (b) 12 cells, and (c) 9 cells.

Figure 4

Graphs showing the effects of thapsigargin (Tg; 100 nM), SKF96365 (20 μM), sodium nitroprusside (SNP; 1 μM) and 8 Br-cyclic GMP (100 μM) on cytoplasmic calcium levels (measured as the Fura-2 ratio; R_340/380) in single smooth muscle cells from the mouse anococcygeus. Cells were bathed initially in calcium-free medium, before exposure to Tg; 2.5 mM calcium (Ca) was then added 10 min after Tg. Each point is the mean±s.e.mean from at least five individual cells.

Figure 5

Graphs showing the time-course of tension changes in mouse anococcygeus muscles skinned with β-escin. Muscles were bathed initially in a calcium-free relaxing solution and then exposed sequentially to calcium ions (Ca²⁺; 1 μM), GTP (100 μM) and then (a) nitric oxide (NO, 96 μM; n=6), b) carbachol (carb, 50 μM) followed by NO (n=6), and (c) thapsigargin (Tg, 100 nM) followed by NO (n=5). Results are expressed as a percentage of the peak contraction produced by calcium alone. Each point is the mean±s.e.mean ^*significant relaxation induced by NO (P<0.05 compared with value immediately before addition of NO).