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 ®eld stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 mM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H- [1,2,4] 4 In b-escin skinned preparations, NO had no e ect on tone induced by calcium (1 mM in the presence of 100 mM 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 re®lling the internal stores, since reduction in [Ca 2+ ] 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.

Receptor‐independent activation of Rho‐kinase‐mediated calcium sensitisation in smooth muscle

“…β ‐escin‐permeabilised, or ‘skinned’, smooth muscles have been used widely to study calcium sensitisation mechanisms, making use of the fact that because the internal milieu of the smooth muscle is a continuum of the extracellular medium in such preparations, the experimenter has some control over the intracellular concentration of calcium ions. As we have reported previously (Ayman et al ., 2001), the addition of calcium to mouse anococcygeus permeabilised with β ‐escin produced concentration‐dependent contractions, with 1 μ M calcium producing approximately 60% of the maximal response. In the present series of experiments, we have used 1 μ M calcium throughout and muscle tension is normalised with respect to that seen in the presence of this concentration of calcium.…”

“…Mouse anococcygeus muscles were dissected and set up initially in Krebs' solution as described above, with the exception that the experiments were performed at 25°C to prolong tissue viability (Ayman et al ., 2001). As before, the Krebs' solution contained phentolamine plus L‐NOARG and the tissues were incubated with guanethidine for 10 min during a 30 min initial equilibration period.…”

“…An early observation that both carbachol (CCh) and Tg can produce strong, well‐maintained contractions in the face of relatively small maintained increases in [Ca 2+ ] i (Wayman et al ., 1999), suggested to us that not only might calcium sensitisation be important in this tissue, but that this process might be activated by both receptor‐dependent and receptor‐independent mechanisms (CCh and Tg, respectively). Recently, we presented preliminary data using β ‐escin‐permeabilised tissues that suggested that calcium sensitisation does indeed occur in this muscle (Ayman et al ., 2001). In the present study, we have extended these observations with the aim of determining first, whether RhoA/Rho‐kinase‐mediated inhibition of SMPP‐1 M is involved in the calcium‐sensitisation response; and second, whether calcium sensitisation can be initiated by receptor‐independent pathways.…”

Nitrergic relaxation in rat gastric fundus: influence of mechanism of induced tone and possible role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

“…This has not yet been evaluated in rat gastric fundus. The exact mechanism by which NO elicits relaxation is still unresolved, but it involves lowering of [Ca 2 + ] i in mouse and rabbit aorta (Cohen et al, 1999), mouse anococcygeus (Ayman et al, 2001) and rat anococcygeus (Raymond and Wendt, 1996). Some reports describe the activation of SERCA as a possible mechanism by which NO induces [Ca 2 + ] i decrease (Petkov and Boev, 1996;Raymond and Wendt, 1996;Wayman et al, 1996a;Cohen et al, 1999), although at least in mouse anococcygeus other mechanisms may also be involved (Ayman et al, 2001).…”

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor‐induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

“…Cyclic GMP may either promote Ca 2+ influx as it does in vertebrate photoreceptors by interacting with cyclic nucleotide gated channels (Stryer 1986) and in rat pituitary cells through dihydro-pyridinesensitive Ca 2+ channels (Willmott et al 1996), or inhibit Ca 2+ influx, particularly in excitable cells (Milbourne and Bygrave 1995). The implication of the NO / cyclic GMP pathway in capacitative Ca 2+ influx is still a controversial issue (Stryer 1986;Ayman et al 2001); however, it appears that in secreting cells the NO / cyclic GMP pathway does promote capacitative Ca 2+ influx through store-operated channels (Xu et al 1994).…”