Dynamic Ca2+ signalling in rat arterial smooth muscle cells under the control of local renin-angiotensin system

Abstract

1. We visualized the changes in intracellular Ca2+ concentration ([Ca2+]i), using fluo-3 as an indicator, in individual smooth muscle cells within intact rat tail artery preparations. 2. On average in about 45 % of the vascular smooth muscle cells we found spontaneous Ca2+ waves and oscillations ( approximately 0.13 Hz), which we refer to here as Ca2+ ripples because the peak amplitude of [Ca2+]i was about one-seventh of that of Ca2+ oscillations evoked by noradrenaline. 3. We also found another pattern of spontaneous Ca2+ transients often in groups of two to three cells. They were rarely observed and are referred to as Ca2+ flashes because their peak amplitude was nearly twice as large as that in noradrenaline-evoked responses. 4. Sympathetic nerve activity was not considered responsible for the Ca2+ ripples, and they were abolished by inhibitors of either the Ca2+ pump in the sarcoplasmic reticulum (cyclopiazonic acid) or phospholipase C (U-73122). 5. Both angiotensin antagonists ([Sar1,Ile8]-angiotensin II and losartan) and an angiotensin converting enzyme inhibitor (captopril) inhibited the Ca2+ ripples. 6. The extracellular Ca2+-dependent tension borne by unstimulated arterial rings was reduced by the angiotensin antagonist by approximately 50 %. 7. These results indicate that the Ca2+ ripples are generated via inositol 1,4, 5-trisphosphate-induced Ca2+ release from the intracellular Ca2+ stores in response to locally produced angiotensin II, which contributes to the maintenance of vascular tone.

Figure 4. Effect of inhibitors of phospholipase C and endothelin on Ca²⁺ ripples

A, Ca²⁺ ripples were abolished by a phospholipase C inhibitor (U-73122, 100 nm, pretreated for 1 min) but not by its inactive analogue (U-73343, 100 nm, pretreated for 1 min). n = 36. B, absence of effect of an endothelin antagonist, PD-142893 (10 μm, pretreated for 5 min). n = 36. Means ±s.e.m.

Figure 5. Effect of an Ang II antagonist, [Sar¹,Ile⁸]-Ang II (10 nm), on Ca²⁺ ripples

A, representative traces of Ca²⁺ ripples before (upper trace), during (middle) and after washout of (lower) [Sar¹,Ile⁸]-Ang II. Ca²⁺ ripples were reversibly abolished. The antagonist was pretreated for 5 min and washed for 2 min before the measurement of [Ca²⁺]_i. B, compiled results of the effect of [Sar¹,Ile⁸]-Ang II on Ca²⁺ ripples (n = 80). P < 0.001. C, effect of a non-peptide AT₁ receptor antagonist, losartan (10 μm), on Ca²⁺ ripples (n = 36). Means ±s.e.m.

Figure 1. Spontaneous Ca²⁺ oscillations (Ca²⁺ ripples) in arterial smooth muscle cells

A, a time-averaged image of fluo-3-loaded smooth muscle cells in isolated strips of the rat tail artery. B, time courses of changes in the fluorescence intensity in small areas (1-7) indicated by □ in A. C, consecutive images of a Ca²⁺ ripple in the cell enclosed by the large box in A. The images were taken at 0.5 s intervals and correspond to the time course enclosed by the box in B, Area 1.

Figure 2. Spontaneous, spike-like increase in [Ca²⁺]_i (Ca²⁺ flash)

A, a time-averaged image of smooth muscle cells in a region where a Ca²⁺ flash was observed. B, intracellular Ca²⁺ images of a Ca²⁺ flash obtained at 0.5 s intervals in a group of three smooth muscle cells enclosed by the large box in A. C, changes in fluorescence intensity plotted against time in the area indicated by the small box (a) in A. D, time courses of changes in the fluorescence intensity of a Ca²⁺ flash enclosed by the box in C on an expanded time scale at 7 equally spaced points within the cell (a-g in A).

Figure 3. Spontaneous Ca²⁺ transients (Ca²⁺ ripples and Ca²⁺ flashes) in comparison with Ca²⁺ transients evoked by NA (30 nm)

A, representative time course of [Ca²⁺]_i before (left) and after (right) application of 30 nm NA. Results from two representative cells that did or did not exhibit Ca²⁺ ripples from two independent arteries (a and b). B, comparison of mean amplitude of Ca²⁺ ripples (156 cells), maximum amplitude in NA-evoked response (142 cells), and mean amplitude of Ca²⁺ flashes (102 cells). Means ±s.d.C, comparison of NA-evoked responses in cells that did or did not exhibit Ca²⁺ ripples. Peak amplitude (left) and the number of peaks in a 100 s observation period (right) are shown. Means ±s.e.m. (n = 20).

Figure 6. Effect of an ACE inhibitor, captopril (10 μm), on Ca²⁺ ripples

Captopril, preincubated for 15 min, inhibited Ca²⁺ ripples in terms of both mean peak amplitude (A) and the number of peaks in a 100 s observation period (B). Means ±s.e.m. (n = 36).

Figure 7. Effect of extrinsic Ang II (100 nm) on [Ca²⁺]_i

A, representative time courses of Ca²⁺ ripples (left) and Ang II-evoked responses (right) in two cells. B, scatter plot of amplitudes of Ang II-evoked responses vs. Ca²⁺ ripples. ○, single samples and • 26 samples. Regression coefficient, 1.028; correlation coefficient, 0.876; P < 0.0001. n = 80. C, comparison of number of peaks in a 100 s observation period between Ca²⁺ ripples and 100 nm Ang II-evoked responses.

Figure 8. Effect of an Ang II antagonist, [Sar¹,Ile⁸]-Ang II (10 nm), on tension in a ring preparation of the tail artery

A, representative tension trace and the effect of [Sar¹,Ile⁸]-Ang II. High potassium solution (80 K) and Ca²⁺-free PSS (Ca²⁺ free) were used. B, compiled results of the effect of [Sar¹,Ile⁸]-Ang II and Ca²⁺-free PSS on tension compared with the tension evoked by a high potassium solution. Means ±s.e.m. (n = 5).