Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Abstract

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.

Figure 1

Spontaneous Ca²⁺ events on endothelial cells of pressurized arteries. (A) Confocal fluorescence image of arterial endothelial cells loaded with Oregon Green 488 BAPTA-1. Scale bar is 30 · m. (B) Time course of fluorescence intensity changes within individual cells shown as ‘line-scans’ in top panels (corresponding to numbered lines shown in A; pseudocolour: blue lowest to red highest) and subcellular regions of interest in bottom panels (F/F₀ in the regions corresponding to the lettered boxes shown in A). ACh (0.3 · M) was added towards the end of the acquisition period, whereupon mixing created an artefact (within the first 10 s). (C) Time course of average fluorescence intensity changes within one cell to illustrate the wave-like nature of the spontaneous event. Coloured boxes in A correspond to the changes in F/F₀. A movie file corresponding to these data is available online (video 1). (D) The distribution of cells displaying spontaneous Ca²⁺ events (Responding cells, top) and the frequency of events in these cells (Hz, bottom) under control condition (n = 99). (E) Reproducibility of Ca²⁺ events (at 0 and 20 min) in the same cell (left) and then at 20 min in another randomly selected cell (right).

Figure 2

Effects of cyclopiazonic acid (CPA), U-73122, ryanodine, and nicotinamide on Ca²⁺ events in endothelial cells of pressurized arteries. CPA (10 · M, n = 3) and U-73122 (1 · M, n = 5) abolished the Ca²⁺ events. Neither ryanodine (20 · M, n = 5) nor nicotinamide (6 mM, n = 3) had a significant effect on spontaneous Ca²⁺ events. Values are means · S.E.M. *P < 0.05, significantly different from control.

Figure 3

Effects of smooth muscle cells on Ca²⁺ events in endothelial cells. (A and B) Stimulation of smooth muscle cells with phenylephrine (PE, 0.9 · 0.2 · M, n = 11) or KCl (34 · 2 mM, n = 11) increased Ca²⁺ events in endothelial cells. (C and D) Application of carbenoxolone (CBX, 100 · M, n = 4) or 18· -glycyrrhetinic acid (18· -GA, 100 · M, n = 4) substantially decreased Ca²⁺ events. The inhibitory effects of CBX (n = 4) continued even in the presence of PE or KCl, but 18· -GA was less effective (n = 4). Values are means · S.E.M. *P < 0.05, significantly different from control.

Figure 4

Simultaneous measurement of Ca²⁺ events in endothelial and smooth muscle cells in rat mesenteric arteries. (A) Confocal fluorescence image of arterial endothelial and smooth muscle cells loaded with Oregon Green 488 BAPTA-1. Scale bar is 30 · m. (B) Time course of fluorescence intensity changes within individual cells shown as subcellular regions of interest (F/F₀ in the regions corresponding to the lettered boxes shown in A). A movie file corresponding to these data is available online (video 2). In these arteries, phenylephrine (PE) significantly increased Ca²⁺ events in both endothelial cells (C) and CWWs in smooth muscle cells (D). Either carbenoxolone (CBX, 100 · M) or U-73122 (1 · M) inhibited Ca²⁺ events in endothelial cells (C) without affecting smooth muscle cells events (D). Values are means · S.E.M. n = 3 for each. *P < 0.05, significantly different from control. ^†P < 0.05, significantly different from CBX without PE.

Figure 5

Effect of carbenoxolone on the loading of calcein into the artery wall. Image z-stacks were captured following selective endothelial cell loading of calcein AM. Confocal images at the level of the endothelial cells (A) and in the same arteries (x- and y-positions unchanged, z-position 4.5 · m down towards the outer wall) at the level of the smooth muscle cells (B). The IEL was stained with Alexa Fluor 633 hydrazide (red). Calcein (green) clearly stained the endothelial cells, with the smooth muscle cells (vertical orientation) faintly visible in the control arteries but not visible in arteries incubated with carbenoxolone. Note that nearly all the bright spots correspond to holes through the internal elastic lamina, see highlighted boxes for comparison. Bar = 50 · m. (C) The average peak fluorescence intensity in endothelial cells (ECs), smooth muscle cells (SMCs), and endothelial cell projections (ECPs) compared to the fluorescence intensity of ECs (% F_EC) in control arteries (n = 4) and those treated with CBX (n = 3). See Section ‘Materials’ for details of analysis. Values are means · S.E.M. *P < 0.05, significantly different from control.

Figure 6

Effects of lowered extracellular Ca²⁺, nifedipine, SKF-96365, NiCl₂, and KB-R 7943. Lower extracellular Ca²⁺ (1 mM) decreased the frequency of Ca²⁺ events compared to control (2 mM Ca²⁺, n = 5). To decrease Ca²⁺ in smooth muscle cells, the arteries were incubated with the voltage-gated Ca²⁺ channel inhibitor, nifedipine. Nifedipine (1 · M, n = 5) decreased Ca²⁺ events in endothelial cells. The frequency of Ca²⁺ events was less in the presence of non-selective inhibitors of Ca²⁺ influx SKF-96365 (n = 5), NiCl₂ (500 · M, n = 3), or KB-R 7943 (3 · M, n = 4). Values are means · S.E.M. *P < 0.05, significantly different from control.