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. 2018 Nov:75:21-29.
doi: 10.1016/j.ceca.2018.08.001. Epub 2018 Aug 7.

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries

Lyudmyla Borysova  1 , Kim A Dora  1 , Christopher J Garland  1 , Theodor Burdyga  2
Affiliations

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries

Lyudmyla Borysova et al. Cell Calcium. 2018 Nov.

Abstract

The role of vascular gap junctions in the conduction of intercellular Ca2+ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 μm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s-1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18β-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.

Keywords: Gap junctions; Intercellular Ca(2+)waves; Mesenteric resistance artery; Spreading vasoconstriction.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Experimental set up for simultaneous measurements of smooth muscle intercellular Ca2+ and force at the downstream (T1) and upstream (T2) ends of the rat mesenteric artery. A, Transmitted light image showing the position of the wires used to radially stretch and measure force at T1 and T2. B, Confocal fluorescence images of an artery loaded with Ca2+ indicator showing a local application via delivery pipette to T1 of fluorescently labelled beads (a), 60 mM KCl (b), and 5 mM caffeine (c). The contraction of the downstream end of the artery (T1) seen as local deflection of the wires evoked by 10 s 60 mM KCl pulse described in A can be seen in Supplement Movie 1. Spatial spread of fluorescently labelled beads described in Ba and Ca2+ signal induced by 10 s 60 mM KCl pulse described in Bb can be seen in Supplement Movie 2.
Fig. 2
Fig. 2
Ca2+ signalling and vasoconstriction induced by 60 mM KCl pulses of different duration applied at T1. A, Transmitted light images of the artery (a) at rest and during (b) local stimulation at T1 with 60 mM KCl 10 s pulse. Note the deflection of the wires. B, Fluorescence images of an artery loaded with Ca2+ indicator, recorded at rest (a) and during local KCl 10 s application at T1 (b-f). Time interval between images is 2 s. C, Superimposed traces of KCl induced Ca2+ signal (top panel) measured in five ROIs shown in Ba and force (bottom panel) recorded at T1 (red trace) and T2 (blue trace) (n=5-7). D, Graph showing time-dependent effects of the local KCl application (1 s, 5 s, 10 s and 20 s duration) at T1 on the amplitude and spatial spread of Ca2+ signal (top panel) measured in five ROIs shown in Ba and force (bottom panel) recorded at T1 (red trace) and T2 (blue trace) (n=5–7). The spatial spread of the Ca2+ signal induced by 60 mM KCl 10 s pulse can be seen in Supplement Movie 3 (For interpretation of the references to colour in the text, the reader is referred to the web version of this article).
Fig. 3
Fig. 3
Profiles of membrane potential and tension during TEA and BayK 8644 application. Patterns of spike action potentials (top traces) and phasic contractions (bottom traces) during exposure of mesenteric artery to 10 mM TEA and 1 μM BayK 8644 (indicated by bar, n=4). A and B (top panel) – action potentials appearing as isolated (single) spike or bursts of spikes, respectively; A and B (bottom panel) – phasic contraction associated with single spike or burst of spikes, respectively.
Fig. 4
Fig. 4
Propagating intercellular Ca2+ wave in the mesenteric artery arcade with intact endothelial layer, evoked by 1 s application of 60 mM KCl in the presence of TEA and BayK 8644. A, Fluorescence images of arterial arcade loaded with Ca2+ indicator, recorded at rest (a) and during local stimulation with KCl applied at T1 of the 3rd order branch (b-e). The interval between displayed images A(a–e) is 600 ms. B, Traces corresponding to Ca2+ signals measured in five ROIs in Aa spaced at 1500 μm interval recorded in the absence (n=3, left panel) and the presence of 10 μM nifedipine (n=3, right panel), respectively. The period of acquisition is indicated by grey bar. C, Average speed of KCl-evoked Ca2+ wave propagation measured between 0–1500 μm, 1500–3000 μm, 3000–4500 μm, and 4500–6000 μm (n=3). The propagating Ca2+ wave described in A–C can be seen in Supplement Movie 4. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article).
Fig. 5
Fig. 5
BayK 8644 alone is not sufficient to enable KCl-mediated propagating responses in denuded arteries. A, Fluorescence images of an artery loaded with Ca2+ indicator and treated with 1 μM BayK 8644, recorded at rest (a) and during 1 s application of 60 mM KCl at T1 in the absence (b) and the presence (c-f) of 10 mM TEA added to the bath with 1 μM BayK 8644 (indicated by bar at B). The interval between displayed images A(c–f) is 700 ms. Note rapid propagation of KCl-induced Ca2+ signal and tension from T1 (red traces) to T2 (blue traces) in the presence of both BayK 8644 and TEA. B, Traces corresponding to Ca2+ signals measured in five ROIs in A (top traces) and force (bottom traces) measured in T1 (red trace) and T2 (blue trace) ends of the artery. The period of acquisition indicated by grey bar. C, left and middle panels (a and b) showing average amplitude of KCl-evoked Ca2+ signal and force recorded at T1 and T2, respectively in the presence of BayK 8644 alone (n=7) and following addition of 10 mM TEA (n=7, expressed as % of peak KCl); C, right panel (c) shows an average speed of KCl-evoked Ca2+ wave and constriction propagation measured between T1 (0 μm) and T2 (3000 μm) (n=7). The local Ca2+ signal and propagating Ca2+ wave induced by 1 s KCl pulse in the presence of BayK 8644 alone and following addition of TEA, respectively, can be seen in Supplement Movie 5. In this movie, the artery responded with a burst of three Ca2+ waves, two of each were initiated at the T1 and the third at the T2 (For interpretation of the references to colour in the text, the reader is referred to the web version of this article).
Fig. 6
Fig. 6
Spontaneous intercellular Ca2+ and mechanical waves induced by TEA in the presence of BayK 8644 in mesenteric arteries with intact endothelial layer. A, Fluorescence images of mesenteric artery loaded with Ca2+ indicator, recorded at rest (a) and following incubation with 1 μM BayK 8644 and the addition of 10 mM TEA (b-d) indicated by bar at B. The interval between displayed images A(b–d) is 600 ms. Note rapid propagation of Ca2+ and mechanical waves from T2 (blue traces) to T1 (red traces). B, Traces corresponding to Ca2+ transients (top traces) measured in five ROIs in A and force (bottom traces) recorded in T2 (blue trace) and T1 (red trace) ends of the artery. The period of acquisition indicated by grey bar. C, left and middle panels (a and b) showing average amplitude of propagating single Ca2+ wave (n=5) and force (n=5, expressed as % of peak KCl) measured at T1 and T2, respectively; C, right panel (c) average speed of spontaneous Ca2+ and mechanical waves propagation measured between T1 (0 μm) and T2 (3000 μm) (n=5). The repetitive propagating Ca2+ waves described in A–C can be seen in Supplement Movie 6 (For interpretation of the references to colour in the text, the reader is referred to the web version of this article).
Fig. 7
Fig. 7
Effect of gap junction uncoupler, 18β-GA on spontaneous propagating intercellular Ca2+ waves and force of mesenteric artery loaded with Ca2+ indicator, in the presence of 10 mM TEA and 1 μM BayK 8644. A, Fluorescent images showing MA at rest (a), during fully propagated intercellular Ca2+ wave (b), and at different time points in the presence of 20 μM 18β-GA (c-d). B, Line-scan plot with respect to time, from SMCs of the whole segment of MA (dashed line indicated in A) showing extremely chaotic asynchronous spontaneous activity in individual SMCs. C, Representative graph of 8 experiments showing changes in force (top panel) and Ca2+ signals recorded in three different ROIs (bottom panels) shown in Aa. Note, ROIs 1 (red) and 2 (blue) show the average Ca2+ signal in a small group of cells, while ROI 3 (green) shows the average Ca2+ signal acquired from the whole area of observation. Spontaneous Ca2+ waves and force described in A–C can be seen in Supplement Movie 7 (For interpretation of the references to colour in the text, the reader is referred to the web version of this article).
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