Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle - PubMed (original) (raw)

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

R Flemming et al. J Physiol. 2002.

Abstract

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca2+ channels activated by depletion of intracellular Ca(2+) stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca2+ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca2+ by inhibition of SR Ca2+-ATPase (SERCA) led to sustained elevation of [Ca2+]i that depended on extracellular Ca2+ and slightly enhanced Mn2+ entry. Enhanced background Ca2+ influx did not explain the raised [Ca2+]i in response to SERCA inhibitors because it had marked gadolinium (Gd3+) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca2+ depletion activated SOCs. Strikingly, SOC-mediated Ca2+ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca2+]i rise being greater than that evoked by 20 mM [K+]o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca2+ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca2+ channels allowing Ca2+ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.

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Figures

Figure 1

Figure 1. Responses of cerebral arteriolar smooth muscle cells to SR Ca2+ depletion

D600 (10 μM) was present in all experiments. A-C, SERCA inhibition with 10 μM CPA caused elevation of [Ca2+]i that depended on extracellular Ca2+. [Ca2+]i is given as the ratio of 345 and 380 nm fura-PE3 signals (_R_345/380). Example experiments in standard (1.5 m

m

Ca2+) bath solution (A) and Ca2+-free bath solution containing 0.4 m

m

EGTA (B). C, summary of data from experiments as shown in A and B. Data are mean ±

s.e.m

. changes in fura-PE3 ratio. For each data point, n ≥ 32 (total N = 14) and 41 (total N = 15) for 1.5 m

m

Ca2+ and Ca2+-free groups, respectively. P < 0.001 for the data point marked ** and all subsequent data points. D, SR Ca2+ depletion slightly enhanced Mn2+ influx. Mn2+ quenching of fura-PE3 (excited at 360 nm) was increased by 10 μM CPA. Mn2+ was applied at 0.5 m

m

in Ca2+-free solution without EGTA. Points are mean ±

s.e.m

. data normalised to the fluorescence intensity before addition of Mn2+. Control group, n/N = 65/13. CPA group, n/N = 45/9. * P < 0.05, ** P < 0.01.

Figure 2

Figure 2. SR Ca2+ depletion induces Gd3+ sensitivity

All experiments included 10 μM D600. A-F, the extracellular solution was either standard (1.5 m

m

Ca2+) or Ca2+-free (0.4 m

m

EGTA). A, no effect of 100 μM Gd3+ in control conditions. Ca2+-free solution reduced [Ca2+]i. B and C, effects of 10 and 100 μM Gd3+ after SR Ca2+ depletion with 10 μM CPA (B) or 0.1 μM TG (C) in Ca2+-free solution. D, mean ±

s.e.m

. 100 μM Gd3+-induced reduction of fura-PE3 ratio in the SR Ca2+-depleted group (as in B, n/N = 20/6) but not in the control (as in A, n/N = 43/10). E, 10 μM CPA-induced sensitivity to 10 μM Gd3+ without any exposure to Ca2+-free solution. F, 10 μM Gd3+ sensitivity, followed by Ca2+-free bath solution, after 1.5 h pre-treatment with 1 μM TG in standard bath solution. G, in a 0.1 μM TG-pre-treated arteriole, effects of 1.5 m

m

Ca2+ and 10 μM Gd3+ in the continuous presence of 100 m

m

K+ solution. H, mean ±

s.e.m

. effects of Gd3+ (10 or 100 μM) as a percentage relative to the fura-PE3 ratio before application of CPA (E) or Ca2+-free bath solution. CPA/0 Ca group: as shown in B for 10 Gd3+ (n/N = 10/3) and 100 Gd3+ (n/N = 20/6). TG/0 Ca group: as shown in C for 10 Gd3+ (n/N = 15/6). CPA/1.5 Ca group: as shown in E for 10 Gd3+ (n/N = 32/8). TG-pre/1.5 Ca group: as shown in F for 10 Gd3+ (n/N = 40/9). TG/100 K group: as shown in G for 10 Gd3+ (n/N = 13/3).

Figure 3

Figure 3. Lack of a SOC-associated contractile response

Images of fura-PE3 fluorescence from an isolated arteriole exposed to 10 μM CPA in the presence of 10 μM D600 (A-D) and an arteriole exposed to 60 m

m

K+ in the absence of D600 (E-H). Images A and E show fluorescence with excitation at 345 nm. The other images are ratios of the 345 nm/380 nm signals on a rainbow scale (blue, low [Ca2+]; red, high [Ca2+]). Images A and B were in the absence of extracellular Ca2+ and image C after the addition of 1.5 m

m

Ca2+. Images E and F were in standard bath solution and image G after addition of 60 m

m

K+. Image D is a merger of images A and C, and H a merger of E and G. Images A-D are for the experiment in Fig. 2_B_. The calibration bar in A is 40 μm and applies to all images. I-L, D600 was excluded. I-K, external diameters of isolated arterioles in standard bath solution at room temperature. In K, standard bath solution contained 0.3 m

m l

-NAME, 10 μM indomethacin, 100 n

m

apamin and 100 n

m

charbydotoxin. L, external diameter of an arteriole in intact pial membrane bathed in artificial CSF containing 0.3 m

m l

-NAME at 37 °C. There was bath application of 10 μM CPA, 1 μM TG, or 60 m

m

K+.

Figure 4

Figure 4. SERCA inhibitors do not affect the Ca2+ sensitivity of contractile proteins

Measurements of the external diameter of β-escin-permeabilised arterioles. A and B, free ionised Ca2+ concentrations in the bath solution are given in μM. A, effect of GTP-γ-S (10 μM) in the presence of 0.1 μM Ca2+. B and C, for a paired experiment carried out on one day, the effects of increasing concentrations of Ca2+ on an arteriole in control conditions (B) and on another arteriole in the presence of 10 μM CPA (C). D and E, mean ±

s.e.m

. diameter of arterioles as a percentage of the initial diameter in the absence of Ca2+, shown for control and 10 μM CPA paired data (n = 11 for each), and control and 1 μM TG paired data (n = 6 for each).

Figure 5

Figure 5. SOC-associated [Ca2+]i rise is not too small to cause contraction

A, Ca2+ dependence of contraction in β-escin-permeabilised arterioles (n = 28). Mean ±

s.e.m

. data as described for control arterioles in Fig. 4_D_ and E. The fitted curve is the Hill equation with a mid-point at 0.59 μM and a slope of 1.2. B-D, effects of elevating the K+ concentration (m

m

) of standard bath solution. D600 was excluded from all solutions. Wortmannin was included during [Ca2+]i measurements. B, external diameter measurement. C, [Ca2+]i indicated by fura-PE3. D, plotted against external K+ concentration, the mean ±

s.e.m

. external diameter as a percentage of that in standard bath solution (n = 7), and changes in fura-PE3 ratio (n/N = 29/8). The superimposed curves are fitted Hill equations with mid-points at 37.3 m

m

K+ (diameter) and 33.5 m

m

K+ (Ca2+). The dashed straight line is the mean change in fura-PE3 ratio (0.0697) in response to CPA in the presence of Cao2+ (from Fig. 1_C_), which is similar in amplitude to the effect of Gd3+ shown in Fig. 2_D_.

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