An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture

Abstract

1. A regenerative calcium wave is an increase in cytosolic free calcium concentration ([Ca2+]i) which extends beyond the stimulated cells without decrement of amplitude, kinetics of [Ca2+]i increase and speed of propagation. 2. The aim of the present study was to test the hypothesis that such a wave could be evoked by bradykinin stimulation and by scraping cultured endothelial cells from porcine coronary arteries. 3. Calcium imaging was performed using the calcium-sensitive dye fura-2. A wound or a delivery of bradykinin to two to three cells on growing clusters of approximately 300 cells caused an increase in [Ca2+]i which was propagated throughout the cluster in a regenerative manner over distances up to 400 micrometer. This wave spread through gap junctions since it was inhibited by the cell uncoupler palmitoleic acid. 4. The same experiments performed in confluent cultures caused a rise in [Ca2+]i which failed to propagate in a regenerative way. The wave propagation probably failed because the confluent cells were less dye coupled than the growing cells. This was confirmed by immunohistology which detected a dramatic decrease in the number of connexin 40 gap junctions in the confluent cultures. 5. The regenerative propagation of the wave was blocked by inhibitors of calcium-induced calcium release (CICR) and phospholipase C (PLC), and by suppression of extracellular calcium, but not by clamping the membrane potential with high-potassium solution. 6. We conclude that regenerative intercellular calcium waves exist in cultured islets but not in confluent cultures of endothelial cells. An increase in [Ca2+]i is not sufficient to trigger a regenerative propagation. The PLC pathway, CICR and extracellular calcium are all necessary for a fully regenerated propagation.

Figure 1. Bradykinin-evoked calcium wave in non-confluent endothelial cell culture

A: top left, fusiform growing endothelial cells forming a cluster after 2 days of primary culture. X corresponds to the area in which the cells were directly stimulated by application of bradykinin (800 nm) and the coloured spots indicate the chosen regions of interest where the variations in [Ca²⁺]_i shown in B were measured. The arrow indicates the direction of perfusion flow. Right, four coloured sequential ratiometric images of [Ca²⁺]_i increase obtained 9, 18, 27 and 51 s following stimulation; a regenerative calcium wave started from the area directly stimulated by bradykinin. Scale bar, 200 μm. B, graph showing the calcium mobilization after bradykinin stimulation; the curves correspond to the variation of [Ca²⁺]_i measured in the regions of interest shown in A. Distance from the stimulation area: 146 μm (^), 266 μm (▵), 412 μm (□), 545 μm (•). The mode of representation of the calcium wave in B was used in subsequent figures.

Figure 2. Cytosolic free calcium mobilization in endothelial cells after bradykinin (800 nm) stimulation

A, role of the age of the culture. The culture was confluent (6 days). ^, cell adjacent to the bradykinin stimulation area; ▵, 65 μm from the stimulation area; □, 83 μm from the stimulation area; •, 122 μm from the stimulation area. B, role of cell coupling. Three-day-old cell cluster in the presence of the gap junction uncoupler palmitoleic acid (50 μm). ^, cell directly stimulated by bradykinin; ▵, 20 μm from the stimulation area; □, 83 μm from the stimulation area; •, 300 μm from the stimulation area. C, role of extracellular calcium. Three-day-old cell cluster in the presence of Krebs solution containing low extracellular calcium (2 mm EGTA; 10⁻¹² M Ca²⁺). ^, 143 μm from the stimulation area; ▵, 256 μm from the stimulation area; □, 363 μm from the stimulation area; •, 421 μm from the stimulation area.

Figure 3. Electrical and chemical cell coupling in cultured endothelial cells

A-C: left, phase contrast micrographs of endothelial cell cultures. The horizontal line at the top of each micrograph is the mark of the scalpel blade used for Lucifer Yellow scrape loading. Middle, fluorescence images of the same areas showing the chemical cell coupling visualized by the diffusion of Lucifer Yellow from the wounded cells. Right, traces representing the current flowing through the syncytium in response to a 5 s, 10 mV change in membrane potential. A, 2- to 3-day-old fusiform growing cell cluster; B, 2- to 3-day-old fusiform growing cell cluster in the presence of the gap junction uncoupler palmitoleic acid; C, 6- to 7-day-old cobblestone-like confluent cell culture.

Figure 4. Immunodetection of connexin 40 in cultured endothelial cells

Staining by antibody to connexin 40 is shown by white spots. A, staining was rarely observed in a 6- to 7-day-old cobblestone-like confluent culture. B, staining of the intercellular boundaries in a 2- to 3-day-old fusiform growing cell culture. Scale bar, 1 μm.

Figure 5. Cytosolic free calcium mobilization in endothelial cells after bradykinin (800 nm) stimulation

A, role of membrane potential. Three-day-old cell cluster in an extracellular solution containing high potassium (120 mm). Distance from the stimulation area: 182 μm (^), 235 μm (▵), 403 μm (□), 541 μm (•). B, role of membrane potential plus extracellular calcium. Two-day-old cell cluster in solution containing high potassium (120 mm) and low calcium (2 mm EGTA; 10⁻¹² M Ca²⁺). Distance from the stimulation area: 203 μm (^), 294 μm (▵), 368 μm (□), 569 μm (•). C, role of CICR. Three-day-old cell culture in solution containing dantrolene (10 μm), an inhibitor of ryanodine receptor-dependent CICR. ^, cell directly stimulated by bradykinin; ▵, 122 μm from the stimulation area; □, 167 μm from the stimulation area; •, 250 μm from the stimulation area.

Figure 6. Cytosolic free calcium mobilization in endothelial cells caused by an ionophore or wounding

A, mobilization after an increase in [Ca²⁺]_i caused by the calcium ionophore 4-bromo A23187 (1.7 μm) in a 2-day-old cluster. ^, cell directly stimulated by the ionophore; ▵, 60 μm from the site of stimulation; □, 125 μm from the site of stimulation; •, 300 μm from the site of stimulation. B, mobilization after an increase in [Ca²⁺]_i caused by a small scrape in a 3-day-old cluster. ^, cell adjacent to the injury; ▵, 126 μm from the site of the injury; □, 262 μm from the site of the injury; •, 498 μm from the site of the injury. C, mobilization after a small scrape in a 3-day-old cluster in the presence of solution containing the aminosteroid U-73122 (10 μm), an inhibitor of PLC. ^, cell adjacent to the wound; ▵, 20 μm from the site of the injury; □, 40 μm from the site of the injury; •, 62 μm from the site of the injury.

Figure 7. Cytosolic free calcium mobilization in endothelial cells

A, mobilization after an increase in [Ca²⁺]_i caused by a small scrape in a 3-day-old cluster in the presence of dantrolene (10 μm), an inhibitor of ryanodine receptor-dependent CICR. ^, cell adjacent to the injury; ▵, 60 μm from the site of the injury; □, 112 μm from the site of the injury; •, 200 μm from the site of the injury. B, mobilization after an increase in [Ca²⁺]_i caused by caffeine (20 mm) in a 3-day-old cluster. ^, cell directly stimulated by caffeine; ▵, adjacent cell; □, 80 μm from the stimulation area; •, 120 μm from the stimulation area.

Figure 8. Maximal [Ca²⁺]_i reached in the stimulated cells in the different experiments

A, after bradykinin stimulation in a 3-day-old cell cluster; B, after bradykinin stimulation in a 7-day-old confluent culture; C, after bradykinin stimulation in the presence of the gap junction uncoupler palmitoleic acid; D, after bradykinin stimulation in a 3-day-old cell cluster in solution containing low calcium (2 mm EGTA; 10⁻¹² M Ca²⁺); E, after bradykinin stimulation in a 3-day-old cell cluster in solution containing high potassium (120 mm); F, after bradykinin stimulation in a 3-day-old cell cluster in solution containing high potassium (120 mm) and low calcium (2 mm EGTA; 10⁻¹² M Ca²⁺); G, after stimulation in a 3-day-old cell cluster caused by the calcium ionophore 4-bromo A23187; H, after a small scrape in a 3-day-old cell cluster; I, after caffeine stimulation in a 3-day-old cell cluster.