Patterns of tonic and oscillatory diameter and global [Ca2+]i changes with agonist stimulation
The mean resting diameter of rat mesenteric arteries pressurised to 50 mmHg used in agonist-induced experiments was 212±4. 2
μm (
n=108). Superfusion of rat mesenteric arteries with 10
μM U46619 (
n=20) induced large vascular constrictions with a change in diameter of 144±7.0
μm. Constrictions to
U46619 were not accompanied by oscillations in vessel diameter and remained tonic in nature over the time of exposure to the vasoconstrictive stimulus (4–5 min;
Figure 1a). Similarly, constrictions to ET-1 (100 n
M; change in diameter of 151+13
μm) were always tonic in nature (
n=5;
Figure 1b). PE (10
μM) caused a large constriction of mesenteric arteries (the change in diameter was 141±3.7
μm (
n=50)). In 28% of cases, a tonic constriction was maintained with 10
μM PE (
Figure 1d). In the remainder of cases, unlike
U46619 stimulation, tissues developed regular oscillations in vessel diameter of mean amplitude 28.9±3.0
μm (22±2.0% of initial change in diameter observed to 10
μM PE). These oscillations were of high frequency (8.0±0.4 oscillations min
−1), took 27±2 s to begin following the addition of PE, and were maintained throughout the period of PE exposure (up to 5 min;
Figure 1e). KCl (60 m
M; n=27), like
U46619 and ET, induced tonic constrictions in diameter of 141±6.5
μm, respectively, not significantly different from the maximum change in diameter observed to 10
μM U46619, 100 n
M ET or 10
μM PE (
Figure 1c). Incubation under resting conditions with 10
μM Nw-nitro-
L-arginine (
L-NNA), an inhibitor of nitric oxide synthase and endothelial-mediated relaxations of high K
+-treated pressurised mesenteric arteries (
Shaw et al., 2000), for 30 min had no effect on the change in diameter to subsequent exposure of
U46619 (constrictions of 124±8.3 and 138±14
μm before and after
L-NNA, respectively,
n=6) or PE (constrictions of 119±15 and 138±23
μm before and after
L-NNA, respectively,
n=3). In the case of PE, all the three vessels exhibited rhythmic contractions to the agonist the frequency or amplitude of which were not significantly altered by
L-NNA.
| Figure 1Simultaneous measurement of global [Ca2+]i and diameter pressurised arteries. Vessel wall [Ca2+]i and intraluminal diameter were continuously recorded as described in the Methods section. Panels (a–d) illustrate (more ...) |
Agonist-induced arterial constrictions were accompanied by elevations of global vessel [Ca2+]i (Figure 1). The elevation in [Ca2+]i was transient in all ET-1- or U46619-stimulated vessels and in three out of five PE-stimulated vessels examined. Therefore, the maintained diameter reductions were accompanied by suprabasal, but submaximal, levels of global [Ca2+]i (29±7.5, 69±2.4 or 81±10.2% of peak for ET-1, U46619 or PE stimulations, respectively; Figure 2). In vessels that, following the initial vasoconstriction, showed vasomotion to PE, the diameter oscillations (of amplitude 40±7.1 μm or 28±3.1% of maximum change in diameter to PE) were also accompanied by oscillations in global [Ca2+]i (52±8.2% of the maximum change in [Ca2+]i).
| Figure 2Summary of global [Ca2+]i and diameter measurements in pressurised arteries. Following stimulation with each receptor agonist ET-1 (panel (a)), U46619 (panel (b)) or PE (panel (c)), the peak [Ca2+]i levels and corresponding (more ...) |
Effects of external stimuli on vessel smooth muscle cellular[Ca2+]i profiles
In a separate series of experiments, we used laser-scanning confocal fluorescent microscopy to examine the nature of [Ca
2+]
i changes in individual smooth muscle cells of pressurised vessels in response to agonist stimulation. Intracellular [Ca
2+] levels in individual neighbouring smooth muscle cells were monitored. In all vessels stimulated with
U46619 (
n=9) or ET-1 (
n=5), there was an initial elevation of [Ca
2+]
i in smooth muscle cells, that was associated with the initial vasoconstriction. This was followed, during tonic diameter reductions, by the onset of periodic cyclical changes of [Ca
2+]
i in individual smooth muscle cells (
n=9 vessels), evident as propagated waves of [Ca
2+]
i (
Figure 3). In vessels stimulated with high K
+ solution, there was no evidence of [Ca
2+]
i wave propagation; [Ca
2+]
i appeared elevated in all smooth muscle cells throughout the period of exposure to high K
+ solution (
n=5 vessels) with relatively little fluctuation in steady state. Propagated [Ca
2+]
i waves were also present in these vessels when tonically activated by PE. A montage of five consecutive images illustrating changes in [Ca
2+]
i of adjacent individual smooth muscle cells in the mid-wall of a pressurised artery during exposure to 10
μM PE is shown in
Figure 3b. Also shown in
Figure 3c is a line plot of the [Ca
2+]
i response of the same individual smooth muscle cell to 10
μM PE and 10
μM U46619. This illustrates similar oscillatory [Ca
2+]
i responses during continued stimulation with
U46619 or PE. Upon examination of at least three individual cells per vessel, the frequency of cellular [Ca
2+]
i oscillations when exposed to maximal doses of agonist were 3.58+0.41 min
−1 for
U46619-stimulated vessels (
n=9), 5.66+0.94 min
−1 for ET-1-stimulated arteries (
n=5) and 4.92+0.49 min
−1 for PE-stimulated arteries (
n=18). In tonically constricted vessels, individual cells exhibited little or no synchronicity in response to
U46619, ET-1 or PE, as illustrated in the continuous line plots of cells from one vessel in
Figure 3e – individual cell [Ca
2+]
i changes occurred out of phase with each other. In 40% of vessels (scanned continuously for at least 1 min), there was evidence of [Ca
2+]
i wave events originating from more than one point in individual cells.
| Figure 3Cellular [Ca2+]i responses of a pressurised mesenteric artery during tonic constrictions. (a) Confocal images of [Ca2+]i levels in individual smooth muscle cells of a pressurised artery at rest and immediately following (more ...) |
In all of the above experiments, we utilised concentrations of agonist that produced maximum constrictions of vessels. In a separate set of experiments, we examined the vessel diameters, and patterns of [Ca2+]i oscillations in individual smooth muscle cells, when exposed to submaximal doses of U46619 or ET-1. Measures of 10−8 M–3 × 10−6 M U46619- or 10−9 M–3 × 10−9 M ET-1-induced vessel constrictions that were 44±6.8% (n=7) or 35±14% (n=6) of the maximum, respectively. Constrictions to these submaximal doses of U46619 or ET-1 resulted in cellular [Ca2+]i oscillations of lower frequency, 1.57±0.62 and 2.91±1.20 min−1, respectively, than compared to maximal doses of agonist, 3.53±1.16 and 6.86±1.52 min−1, respectively (two-way ANOVA, P<0.01). An example of vessel constrictions, and the accompanying line plots of [Ca2+]i oscillations from individual cells of the artery, to these submaximal concentrations or maximal doses of ET-1 is shown in Figure 4.
| Figure 4Dose-dependent changes in vessel diameter and in smooth muscle cell [Ca2+]i oscillations. Vessels were constricted with submaximal doses of ET-1 (10−9–3 × 10−9 M) and alterations in diameter and smooth muscle (more ...) |
In pressurised arteries showing oscillations in diameter, there were also periodic [Ca2+]i oscillations noted in individual smooth muscle cells. It has previously been shown that during vasomotion individual cellular [Ca2+]i transients may become synchronised between groups of cells to produce phasic contractions of the whole vessel (Mauban et al., 2001; Peng et al., 2001). Therefore, we examined in detail in Figure 5 how a group of several smooth muscle cells behaved in an artery exhibiting vasomotion. In panel (a), there are two images of the artery showing the positions of the vessel (left) and the cells from which measurements were taken (right). Measurements of fluorescence were taken from ROI in each of the cells as indicated, the [Ca2+]i transients from each were normalised for amplitude and are superimposed in panel (b) (the colour of the trace refers to an individual cell). This normalisation procedure assumes that the rise in [Ca2+]i in each cell is of a similar magnitude. Panel (b) shows that the Ca transients are not synchronous in the different cells, for example, between the two [Ca2+]i transients of adjacent cells marked *, there is a time lapse of 7 s even though they are part of the same ‘cycle' of vasomotion. In the upper part of panel (c), we show the mean of the [Ca2+]i signals from the cells in (b). This averaged [Ca2+]i trace shows phasic increases and decreases that are in phase with the movements of the vessel wall, as shown in the lower trace. Wall movement was tracked using a nonmuscle fluorescent element of the adventitia of the vessel. Panel (c), therefore, illustrates that, during vasomotion of the vessel wall, the average Ca signal from the smooth muscle cells changed in parallel without absolute synchronisation of the [Ca2+]i signals between individual cells. Similar results were seen in another three vessels.
| Figure 5Smooth muscle subcellular Ca changes in a pressurised artery exhibiting vasomotion to stimulation with PE (10 μM). Panel (a), an illustration of [Ca2+]i signals in smooth muscle cells with PE stimulation (left) and outlines of (more ...) |
Agonist-dependent Ca2+ sensitisation of vessel constrictions
The functional response of arteries to [Ca
2+]
i elevations in individual smooth muscle cells will be determined not only by the nature of those [Ca
2+]
i oscillations, but also by any agonist-mediated sensitisation to the activating [Ca
2+]
i. As indicated in the global [Ca
2+]
i measurements above, tonic diameter reductions to each agonist were associated with [Ca
2+]
i levels maintained below the initial peak value indicative of sensitisation of tone. We, therefore, examined further the degree of Ca
2+ sensitisation of contractility induced by each agonist in arteries permeabilised with
α-toxin as, in this situation, the [Ca
2+]
i of the bathing medium surrounding the myofilaments can be clamped while other receptor-coupled signalling events remain intact. In these tissues, exposure to a pCa6.7 GTP-containing solution gave submaximal constrictions 49±6.4% of those to pCa4.5 (
n=12). Subsequent exposure to 10
μM U46619 further promoted vessel constriction such that
U46619-induced Ca
2+-sensitisation was 63±10% of constrictions to pCa4.5 (
n=6). This was significantly greater than the corresponding Ca
2+-sensitisation induced by PE (36±6.8% magnitude of constrictions to pCa4.5;
Figure 6a). In a separate set of experiments, we also compared the ET-1-induced Ca
2+-sensitisation to that of PE. In this case, ET-1-induced Ca
2+-sensitisations of amplitude 52±6.8% of constrictions to pCa4.5, again, significantly greater than those to PE (11±3.1%;
n=6;
Figure 6b).
| Figure 6Comparison of the Ca2+-sensitising actions of U46619 or ET-1 to PE in pressurised mesenteric arteries. (a) Following α-toxin permeabilisation, submaximal force to pCa6.7, in the presence of GTP, was substantially augmented by 10 μM U46619 (more ...) |