Mechanisms of Ca2+ sensitization of force production by noradrenaline in rat mesenteric small arteries

Abstract

1. Mechanisms of Ca2+ sensitization of force production by noradrenaline were investigated by measuring contractile responses, intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of the myosin light chain (MLC) in intact and alpha-toxin-permeabilized rat mesenteric small arteries. 2. The effects of noradrenaline were investigated at constant membrane potential by comparing fully depolarized intact arteries in the absence and presence of noradrenaline. Contractile responses to K-PSS (125 mM K+) and NA-K-PSS (K-PSS + 10 microM noradrenaline) were titrated to 30 and 75%, respectively, of control force, by adjusting extracellular Ca2+ ([Ca2+]o). At both force levels, [Ca2+]i was substantially lower with NA-K-PSS than with K-PSS. With K-PSS, the proportion of MLC phosphorylated (approximately 30%) was similar at 30 and 75% of control force; with NA-K-PSS, MLC phosphorylation was greater at the higher force level (40 vs. 34%). 3. In alpha-toxin-permeabilized arteries, the force response to 1 microM Ca2+ was increased by 10 microM noradrenaline, and MLC phosphorylation was increased from 35 to 45%. The protein kinase C (PKC) inhibitor calphostin C (100 nM) abolished the noradrenaline-induced increase in MLC phosphorylation and contractile response, without affecting the contraction in response to Ca2+. Treatment with ATP gamma S in the presence of the MLC kinase inhibitor ML-9 increased the sensitivity to Ca2+ and abolished the response to noradrenaline. 4. The present results show that that in rat mesenteric small arteries noradrenaline-induced Ca2+ sensitization is associated with an increased proportion of phosphorylated MLC. The results are consistent with a decreased MLC phosphatase activity mediated through PKC. Furthermore, while MLC phosphorylation is a requirement for force production, the results show that other factors are also involved in force regulation.

Figure 1. Force and [Ca²⁺]_i measured simultaneously in response to [Ca²⁺]_o

A, force relative to control force, F₁, in response to cumulative increasing [Ca²⁺]_o in K-PSS (^, 125 mM K⁺) and NA-K-PSS (▪, K-PSS plus 10 μM noradrenaline) in intact rat mesenteric small arteries. B, from the same experiments; simultaneous measurements of [Ca²⁺]_i using fura-2 plotted against [Ca²⁺]_o. The [Ca²⁺]_i recorded is the mean concentration in the last 20 s of a stimulation period of 3 min. Error bars indicate s.e.m. (n = 4).

Figure 2. Time course of the Ca²⁺-sensitizing effect of noradrenaline investigated by simultaneous measurements of [Ca²⁺]_i and contractile response in intact rat mesenteric small arteries

A and B, original traces showing contractile responses (upper) and [Ca²⁺]_i (lower) measured in arteries mounted in a myograph under isometric conditions. In A, the artery was stimulated with K-PSS for 20 min. In B, the artery was stimulated with NA-K-PSS. C, readings at 2, 5 and 20 min of simultaneously measured contractile responses (left panel, measured relative to control force, F₁) and [Ca²⁺]_i (right panel). Arteries were stimulated with K-PSS (open bars) or with NA-K-PSS (hatched bars). Error bars indicate s.e.m. (n = 7). *P < 0.05, **P < 0.01, using a Student's paired t test to test the difference between responses to K-PSS and NA-K-PSS at each time point.

Figure 3. Time course of the Ca²⁺-sensitizing effect of noradrenaline investigated by simultaneously measured MLC phosphorylation and contractile response in intact rat mesenteric small arteries

A, Western blot of homogenates of arteries which had been mounted in an isometric myograph and quick-frozen under the conditions indicated, i.e. relaxation conditions (PSS), after stimulation with K-PSS for 5 min, and after stimulation with NA-K-PSS for 5 min. Arterial proteins were extracted, separated by urea-glycerol gel electrophoresis, blotted onto a PVDF membrane, labelled with MLC antibodies and detected by a chemiluminescence detection system. Each lane contains proteins from two 3 mm segments mounted in the same double myograph. The upper band is the unphosphorylated MLC and the lower band is the monophosphorylated MLC. Each panel represents double determinations. The arrow indicates the direction of migration. B, developed force (left) measured relative to control force, F₁, and the fraction of phosphorylated MLC (right) of arteries frozen 2, 5 and 20 min after start of stimulation with K-PSS (125 mM K⁺) (open bars) or NA-K-PSS (hatched bars). Error bars indicate s.e.m. (n = 4–6). **P < 0.01, using a Student's unpaired t test to test the difference between responses to K-PSS and NA-K-PSS at each time point.

Figure 4. Effect of noradrenaline on the relation between MLC phosphorylation and force in intact rat mesenteric small arteries

Arteries were depleted for Ca²⁺ in intracellular stores and stimulated with K-PSS (open symbols) or NA-K-PSS (filled symbols) with variable [Ca²⁺]_o as indicated. The upper graphs show, as functions of [Ca²⁺]_o (μM), force development (A) and phosphorylation (B) determined after 5 min of stimulation, where [Ca²⁺]_o was adjusted to give either 30 % (triangles) or 75 % (squares) force relative to control force, F₁ (see Methods). C, same data plotting force versus MLC phosphorylation. , non-stimulated arteries (held in PSS) (n = 2); •, arteries stimulated for 5 min with NA-K-PSS, both with high [Ca²⁺]_o (2·5 mM) (n = 2). Continuous lines connect paired data (n = 12), where the arteries were mounted on two double myographs run in parallel. Dashed lines indicate tentative curves connecting paired K-PSS or NA-K-PSS data with minimum and maximum values. Error bars indicate s.e.m. where this exceeds the size of the symbol.

Figure 5. Force responses of permeabilized rat mesenteric small arteries to the permeabilization procedure and to subsequent application of Ca²⁺

A, traces show the response of an artery to permeabilization with α-toxin in the presence of 1 μM Ca²⁺, then to cumulative applications of Ca²⁺, and then a repeat of the latter 80 min later. The permeabilization was performed (left traces) with 1000 U ml⁻¹α-toxin in the presence of 1 μM Ca²⁺ until the force appeared to be close to its maximum. In centre and right traces, Ca²⁺ was applied with increasing concentrations at intervals of 3 min: A, 115 nM; B, 317 nM; C: 573 nM; D, 737 nM; E, 955 nM. In F, 10 μM noradrenaline was added in addition to 955 nM Ca²⁺ for 5 min. During the break in the trace (80 min), the artery was stimulated twice with 955 nM Ca². In the lower trace, 6 μM GTP was present during the time indicated by the bar. In the upper trace, no exogenous GTP was present in the bath at any time. The left panel in B (early) shows Ca²⁺ response curves made just after the permeabilization in the presence of different GTP concentrations: 0 μM (^), 2 μM (□), 4 μM (▵), 6 μM (•). The right panel (late) shows the second Ca²⁺ response curves, as described in A, repeated with the respective GTP concentrations (identified as in left panel). Error bars indicate s.e.m.

Figure 6. Effect of GDPβS on the contractile response to 1 μM Ca²⁺ in permeabilized rat mesenteric small arteries

Trace shows 100 μM GDPβS added in addition to stimulation with 1 μM Ca²⁺ 20 min after permeabilization. No exogenous GTP was present. Trace is representative of 3 experiments.

Figure 7. Inhibition of noradrenaline-induced Ca²⁺ sensitization in permeabilized rat mesenteric small arteries with calphostin C

A, traces show 40 min stimulation with 1 μM Ca²⁺ in a permeabilized artery (left), and similar stimulation with 1 μM Ca²⁺, but with 10 μM noradrenaline (NA) added at 10 min (right). B, as A but in the presence of 100 nM calphostin C (after 1 h pre-incubation).

Figure 8. Thiophosphorylation in the presence of ML-9 increases the response of rat mesenteric small arteries to Ca²⁺

A, permeabilized arteries were treated with ML-9 and ATPγS as described by Trinkle-Mulcahy et al. (1995). The increased Ca²⁺ sensitivity following this treatment is shown by a comparison of the stimulations with 1 μM Ca²⁺ before and after treatment. B, detection of proteins labelled with [³⁵S]ATPγS (see Methods) in the presence of ML-9 and absence of Ca²⁺. Following the protocol shown in A, but with 50 μM GTPγS as well as [³⁵S]ATPγS, arteries (3 mm long) were frozen at the point labelled ‘X’, and total protein was extracted, separated by gel electrophoresis (4–20 % SDS-PAGE) and prepared for autoradiography. The arrow indicates direction of migration. The molecular masses of the three major bands and the expected position of MLC (20 kDa) is shown on the right. The gel is typical of three experiments.

Figure 9. Thiophosphorylation in the presence of ML-9 inhibits noradrenaline-induced Ca²⁺ sensitization of rat mesenteric small arteries

Traces are from two arteries treated with ATPγS-ML-9 and stimulated with 0.15 μM Ca²⁺ followed by stimulation with 1 μM Ca²⁺, giving near-maximal contraction. A, control. B, noradrenaline was added on top of stimulation with 0.15 μM Ca²⁺ as indicated; note the small size of the response to noradrenaline (NA). The traces are representative for four experiments. In some of these (as here), ATP was omitted from the relaxing solution during thiophosphorylation.