Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries

Abstract

The present study was aimed at investigating whether, besides its pivotal role in Ca(2+)-independent contraction of smooth muscle, Rho-kinase is involved in the mechanisms underlying the Ca2+ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho-kinase inhibitor Y-27632 (10 microM) completely relaxed the contraction evoked by noradrenaline (1 microM) and simultaneously inhibited the Ca2+ signal by 54 +/- 1 % (mesenteric artery) and 71 +/- 15 % (aorta), and the cell membrane depolarisation by 56 +/- 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF4-, while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca2+. The same effects were observed with another inhibitor of Rho-kinase (HA1077) but not with an inhibitor of protein kinase C (Ro-31-8220). Effects of Y-27632 were not prevented by incubating the artery in 25 mM KCl, with K+ channel blockers or with the Ca2+ channel blocker nimodipine. Y-27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca2+ from non-mitochondrial stores evoked by InsP3 in permeabilised aortic cells, or the Ca2+ signals evoked by thapsigargin or caffeine. The capacitative Ca2+ entry activated by thapsigargin was not impaired by Y-27632, but the entry of Ba2+ activated by noradrenaline in the presence of nimodipine was blocked by 10 microM Y-27632. These results indicate that Rho-kinase is involved in noradrenaline activation of a Ca2+ entry distinct from voltage- or store-operated channels in rat arteries.

Figure 2. Effect of Y-27632 on noradrenaline concentration-response curves in fura-2-loaded mesenteric artery (A and B) and aorta (C and D)

[Ca²⁺]_cyt (upper traces and graphs) and contraction (lower traces and graphs) were simultaneously measured in control artery rings or in artery rings pre-incubated for 10 min in the presence of 10 μM Y-27632. A and C, typical traces obtained in untreated and Y-27632-treated segments from the same artery are superimposed. B and D, mean values from 4–5 different arteries. Vertical bars represent the s.e.m. values.

Figure 1. Representative recordings of the effect of Y-27632 on the Ca²⁺ signal and the contraction evoked by noradrenaline (A), AlF₄⁻ (B) or 100 mM KCl (C) in rat mesenteric artery rings

[Ca²⁺]_cyt (upper trace) and contraction (lower trace) were measured simultaneously in fura-2-loaded arteries. Traces A, B and C are from different arteries. Noradrenaline (NA, 1 μM), AlF₄⁻ (30 μM), KCl (100 mM) and Y-27632 (10 μM) were applied as indicated.

Figure 3. Representative recordings of the effect of HA1077 and Ro-31–8220 on Ca²⁺ signal (upper traces) and contraction (lower traces) evoked by noradrenaline in fura-2-loaded aorta

Noradrenaline (NA, 1 μM), HA1077 (10 μM), Ro-31–8220 (3 μM) and Y-27632 (10 μM) were applied as indicated.

Figure 4. Concentration-inhibition curves for effects of Y-27632 on the Ca²⁺ signal and the contraction evoked by noradrenaline in mesenteric artery

Fura-2-loaded arteries were incubated with Y-27632 (10 μM) for 15 min before stimulation with 1 μM noradrenaline. Responses were expressed as a percentage of the control responses recorded before the incubation with Y-27632. Each point is the mean value of 4–5 determinations. Vertical bars represent the s.e.m. values.

Figure 5. Effect of Y-27632 on the depolarisation evoked by noradrenaline in mesenteric artery smooth muscle cells

Representative recording of the change in membrane potential (upper trace) and in tension (lower trace) evoked by noradrenaline (NA, 1 μM) in mesenteric artery before and after incubation with Y-27632 (10 μM). Traces are from the same artery segment.

Figure 6. Effect of Y-27632 on InsP₃-evoked ⁴⁵Ca²⁺ release in permeabilised A7r5 aortic cells

A, ⁴⁵Ca²⁺-loaded Ca²⁺ stores were allowed to release Ca²⁺ passively in the efflux medium with or without Y-27632 (10 μM). InsP₃ (0.3 and 1 μM) was applied for 2 min as indicated by the horizontal bar. Ca²⁺ release was plotted as fractional release (the amount of Ca²⁺ leaving the stores in 2 min divided by the total store content at that time). Points are means from four experiments. B, Ca²⁺ release evoked by a 2 min application of various concentrations of InsP₃ in the absence or presence of 10 μM Y-27632.

Figure 7. Inhibition of the responses to noradrenaline by Y-27632 in nimodipine-treated aorta

Representative recording of the effect of noradrenaline (1 μM NA) on [Ca²⁺]_cyt (upper trace) and contractile tension (lower trace) in fura-2-loaded aortic segments pretreated with the Ca²⁺ channel blocker nimodipine (1 μM). Y-27632 (10 μM) or vehicle were added at the time indicated by the dashed arrow. Control and Y-27632 traces were from different segments of the same aorta.

Figure 9. Effect of Y-27632 on the different components of the Ca²⁺ signal evoked by noradrenaline in fura-2-loaded aorta in the absence or presence of nimodipine

Columns represent the mean values of the increase in F₃₄₀/F₃₈₀ ratio measured in Ca²⁺-free solution at the time of the peak Ca²⁺ response to noradrenaline (1 μM) (A), or after re-addition of Ca²⁺ (1.25 mM) to the Ca²⁺-free solution (B), in untreated aorta, or in aorta pretreated with Y-27632 (10 μM), nimodipine (1 μM), or nimodipine plus Y-27632. The protocol of the experiment was as represented in Fig. 8A. The effect of re-addition of Ca²⁺ was measured with reference to the baseline fluorescence ratio measured in Ca²⁺-containing solution. Increases in the F₃₄₀/F₃₈₀ ratio were expressed as a percentage of the basal F₃₄₀/F₃₈₀ value and are means from 7–13 determinations ±s.e.m.*P < 0.05.

Figure 8. Effect of Y-27632 on the Ca²⁺ signal and the contraction evoked by noradrenaline in aorta in Ca²⁺-free solution

A, representative recording of the effect of pre-incubation of fura-2-loaded aorta with Y-27632 on the Ca²⁺ signal (upper traces) and contraction (lower traces). Physiological solution was changed to Ca²⁺-free solution as indicated by the horizontal line. Noradrenaline (NA, 1 μM) was applied as indicated by the arrow. Traces obtained before and after incubation with Y-27632 (10 μM) are superimposed. They were obtained from the same artery ring. B, representative recording of Ca²⁺ signal (upper trace) and contraction (lower trace) showing the effect of Y-27632 (10 μM) applied during the re-addition of Ca²⁺ to the perfusion solution in a fura-2-loaded aorta pretreated with nimodipine (1 μM). Noradrenaline (NA, 1 μM) and Y-27632 (10 μM) were applied as indicated.

Figure 10. Effect of Y-27632 on Ba²⁺ entry in fura-2-loaded aorta

A, superimposed representative recordings of the changes in the F₃₄₀/F₃₈₀ ratio evoked by the addition of Ba²⁺ (1 mM) into nimodipine-containing Ca²⁺-free perfusion solution. Vehicle (control) or Y-27632 (10 μM) was added at the time indicated by the dashed arrow. Traces were from different aortas. B, superimposed representative recordings of the changes in F₃₄₀/F₃₈₀ ratio evoked by the addition of Ba²⁺ (1 mM) to nimodipine-containing Ca²⁺-free perfusion solution after stimulation with 1 μM noradrenaline (NA). Vehicle (control) or Y-27632 (10 μM) was added at the time indicated by the dashed arrow. Traces were from different aortas. C, bar graph showing the mean values of the change in the F₃₄₀/F₃₈₀ ratio evoked by Ba²⁺ expressed as a percentage of the basal F₃₄₀/F₃₈₀ value in unstimulated aorta (n = 6) and in aorta stimulated by 1 μM noradrenaline (NA, n = 7) in the absence and presence of Y-27632 (10 μM). D, bar graph showing the mean values of the rate of Ba²⁺ entry expressed as change in F₃₄₀/F₃₈₀ ratio s⁻¹ in unstimulated aorta (n = 6) or in aorta stimulated by 1 μM noradrenaline (NA, n = 7) in the absence (□) and presence of Y-27632 (10 μM) (□). *P < 0.05.

Figure 11. Effect of Y-27632 on the Ca²⁺ signal evoked by caffeine or thapsigargin in fura-2-loaded aorta

A, representative recording of the effect of pre-incubation of the aorta with Y-27632 (10 μM) on the Ca²⁺ signal evoked by caffeine. Noradrenaline (NA, 1 μM) was applied 3 min before stimulation with caffeine (Caf, 10 mM). Traces obtained before and after incubation with Y-27632 are superimposed. They were obtained from the same aortic ring. B, bar graph showing mean values of the Ca²⁺ signal evoked by noradrenaline (NA); caffeine measured at the peak (Caf peak) and the plateau of the response (Caf plat) in control (□) and in Y-27632-treated aorta (□). Data were normalised to the response to 100 mM KCl and are means from 4 determinations ±s.e.m.*P < 0.05, Y-27632-treated vs. untreated aortas. C, representative recording of the effect of Y-27632 on Ca²⁺ signal evoked by thapsigargin. Thapsigargin (Tg, 1 μM) and noradrenaline (NA, 1 μM) were applied as indicated by the arrows. The physiological solution was changed to a Ca²⁺-free solution as indicated by the horizontal bar. Traces obtained without and with Y-27632 (10 μM) are superimposed. They were obtained from different aortic rings. D, bar graph showing mean values of Ca²⁺ signal evoked by thapsigargin (Tg), noradrenaline (NA) measured at the peak of the response in Ca²⁺-free solution, and after re-admission of Ca²⁺ (1.25 mM) to the solution (+Ca) in control (□) and in Y-27632-treated aorta (□). Effect of re-addition of Ca²⁺ was measured with reference to the baseline fluorescence ratio measured in Ca²⁺-containing solution. Data were normalised to the response to 100 mM KCl and are means from 4 determinations ± S.E.M.