Functional Tuning of Intrinsic Endothelial Ca2+ Dynamics in Swine Coronary Arteries

Abstract

Rationale: Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca(2+) transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca(2+) is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca(2+) signals in the coronary endothelium is poorly understood.

Objective: We aimed to determine whether a discrete spatial and temporal profile of Ca(2+) dynamics underlies endothelium-dependent relaxation of swine coronary arteries.

Methods and results: Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca(2+) dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca(2+). In vessel rings, inhibition of specific Ca(2+)-dependent endothelial effectors, namely, small and intermediate conductance K(+) channels (K(Ca)3.1 and K(Ca)2.3) and endothelial nitric oxide synthase, produces additive tone, which is blunted by internal store depletion or inositol 1,4,5-trisphosphate receptor blockade. Stimulation of endothelial inositol 1,4,5-trisphosphate-dependent signaling with substance P causes idiosyncratic changes in dynamic Ca(2+) signal parameters (active sites, event frequency, amplitude, duration, and spatial spread). Overall, substance P-induced vasorelaxation corresponded poorly with whole-field endothelial Ca(2+) measurements but corresponded precisely with the concentration-dependent change in Ca(2+) dynamics (linearly translated composite of dynamic parameters).

Conclusions: Our findings show that endothelium-dependent control of swine coronary artery tone is determined by spatial and temporal titration of inherent endothelial Ca(2+) dynamics that are not represented by tissue-level averaged Ca(2+) changes.

Keywords: calcium signaling; coronary vessels; myography; potassium channels; substance P.

Figure 1. Basal dynamic Ca²⁺ events in swine coronary artery endothelium

A, Ca²⁺-dependent fluorescence recorded from the intimal surface of opened Fluo-4 AM loaded artery segments (Bar = 20 μm). The gray-scale image (left) shows an average-intensity projection over a 10-minute confocal recording. Regions of Ca²⁺ change over the full recording were autodetected by the custom software LC_Pro and displayed as a binary mask (Right; Ca²⁺ events in black). Individual events occurring over the time course are shown below. B, Time course (intensity dependent pseudocolor) of typical basal Ca²⁺ events, including localized transients (top) and a cellular wave (bottom). For each, embedded pseudo-line scan analysis shows continual time course along the dotted line. Arrows indicate initiation of the wave event. Right panel shows a maximal intensity projection of Ca²⁺ (over 30 seconds) in a cell exhibiting both localized events and waves.

Figure 2. Quantification of basal endothelial Ca²⁺ signals

A, Recording shows Ca²⁺ signals autodetected during the 10-minute sample shown in Fig 1A (red line depicts average signal over the field). Insets show three disparate basal Ca²⁺ events. B, Histograms summarizing basal Ca²⁺ event parameters. Data were acquired from 8 animals.

Figure 3. Dependence of basal endothelial Ca²⁺ events on endoplasmic reticulum IP₃ receptors (IP₃Rs)

A, Recordings show endothelial Ca²⁺ signals recorded in opened coronary arteries before and after the perturbations or drug additions indicated (15 minute pretreatment). B, Bar graph summarizes net change in the occurrence of Ca²⁺ events after the indicated treatments CPA, cyclopiazonic acid; Ry, ryanodine; Xest C, xestospongion C; n=4–5, *p<0.01 versus Ca²⁺ free). C, Immunostaining for IP₃R (red) shows distinct clustering around endothelial cell nuclei and down the cell axis; nuclei (green). Bar = 20 μm. Volume render of merged 10-μm image z-stack (below). D, Evaluation of IP₃R expression and Ca²⁺ within a selected cell following a basal Ca²⁺ wave. Arrows indicate spatial correspondence of high and low Ca²⁺ signals with relative IP₃R density; N, nucleus.

Figure 4. Contribution of eNOS, K_Ca2.3, and K_Ca3.1 channels to persistent endothelial modulation of coronary artery tone

A, The representative myograph recording shows successive contractions of isolated coronary artery rings solicited by serial additions of LNNA (200 μmol/L), apamin (Apa; 0.5 μmol/L) and charybdotoxin (Chtx; 0.1 μmol/L). Net contraction was not further enhanced by 60 mmol/L KCl. For all vessel studied, low concentrations of U46619 (1 – 10 nmol/L) were used to standardize resting tone at 7 mN. B, Summary of LNNA, Apa, and Chtx contractions in the presence (+Endo) or absence (−Endo) of endothelium (n=20 and 11). C, Immunostaining for eNOS (red, left) or K_Ca3.1 (green, right) and K_Ca2.3 (red, right) channels in open swine coronary artery (bar = 20 μm). Nuclei are stained blue. Positive staining was detected in the endothelium (E) but not underlying smooth muscle (VSM). Inset (bottom) shows co-labeling of Golgi apparatus (G; red) and eNOS (eN; green) in densities near the nucleus (blue). D, Tracking both Ca²⁺ (Fluo-4 AM) and NO (DAR-4M AM) in an endothelial cell shows the spatial and temporal relationship between basally occurring Ca²⁺ events and NO production (both in pseudocolor). Dotted line shows position of pseudo-line scan (lower left) and white squares indicate the cell region sampled in the associated recording (lower left). Corresponding recordings of whole-cell Ca²⁺ and NO signals are shown on the right.

Figure 5. Role of IP₃ signaling in eNOS and K_Ca channel dependent modulation of coronary artery tone

A, Bar graphs show the impact of various pretreatments (15 min) on LNNA (left) or Apa and Chtx-induced (right) contractions. Pretreatments included the RyR inhibitor ryanodine (Ry, 10 μmol/L), IP₃R blockers xestospongin C (Xesto, 30 μmol/L) and 2-APB (100 μmol/L), the PLC inhibitor U73122 (10 μmol/L) and the nonselective cation channel inhibitor Gd³⁺ (100 μmol/L); n = 5–7, * indicates p<0.05. B, Representative myograph recordings show differential impacts of ryanodine and xestospongin C pretreatments on Apa, Chtx and LNNA contractions.

Figure 6. Effects of endothelial agonist substance P on arterial tone and endothelial Ca²⁺ signaling

A, Representative myograph recordings show concentration-dependent substance P (SP) relaxations and impaired relaxations in arteries pretreated with U73122 or the combination of LNNA, Apa and Chtx. All arteries were pre-contracted to 50–70% of maximal tone with U46619 (10–100 nmol/L). B, Representative recordings of Ca²⁺-dependent fluorescence in open coronary artery preparations show impacts of increasing concentrations of SP on discrete Ca²⁺ dynamics. Red lines indicate Ca²⁺ levels over the whole sampled fields. Insets (right) show cumulative masks of active Ca²⁺ sites (black) before and after SP addition (180 sec each).

Figure 7. Summary of concentration-dependent substance P (SP)-induced changes in endothelial Ca²⁺ event parameters

Data represent cumulative assessment of five experiments per SP concentration. * indicates p< 0.05 vs. 10⁻¹³ μmol/L; # indicates p< 0.05 vs. 10⁻¹³ μmol/L for events.

Figure 8. Correspondence of substance P (SP) relaxation with endothelial Ca²⁺ dynamics

A, Left: Changes in individual dynamic Ca²⁺ parameters (black lines) are plotted as a function of SP concentration (symbols are mean values). Also plotted is the mean whole-field Ca²⁺ change within the same sampled fields (red line). Right: Ca²⁺ dynamics are combined into one curve representing the average of all parameters at each SP concentration. B, Curves show Ca²⁺ dynamics and whole field Ca²⁺ (same data in A) expressed as percent max and plotted alongside concentration-dependent SP arterial relaxation (n=5–6 for each concentration).