Calcium dynamics and intercellular communication in arterial smooth muscle cells

This thesis is a contribution to the field of cellular communication in arteries during vasomotion, i.e. rhythmic and spontaneous diameter oscillations of arteries. Investigating how individual smooth muscle cells (SMCs) and endothelial cells (ECs) calcium variations interact to induce an arterial response is a key to understanding the physical mechanisms leading to contraction and vasomotion. This study is composed of two main experimental parts using rat mesenteric arteries: a study on recruitment and synchronization of SMCs and an analysis on SMCs-ECs communication in arterial strips. The introduction is an overview of the mechanism leading to arterial vasomotion. We focused on the calcium signaling between SMCs, between ECs and between SMCs and ECs. In the chapter "Material and methods", we present the different experimental techniques we developed or improved in order to study calcium signaling in SMCs and/or ECs. For this purpose, we used arterial strips or cannulated arteries using a confocal microscope or a conventional microscope. Calcium concentration were measured using fluorescent dyes. These methods allow the correlation of calcium oscillations of individual SMCs, of individual ECs or both, together with mean calcium variations and arterial contraction. In chapter 3, we investigated the behavior of individual SMCs in order to determine if all cells presented the same variations of calcium concentration as the mean calcium variations or if vasomotion results of an unequal contribution of each SMC. Arterial strips were stimulated by increasing concentration of vasoconstrictors phenylephrine (PE) or potassium chloride (KCl). We revealed that the number of SMCs presenting calcium variations and their synchronization depend on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells present asynchronous calcium variations and no local contraction is detected. Recruitment of cells is complete and synchronous at medium concentration, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous oscillations and a fully contracted arterial strip, whereas high concentration of KCl leads to a sustained non-oscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling artery contraction and vasomotion. In chapter 4, we investigated the three main possible ways to synchronize the recruitment of SMCs when stimulating with PE and KCl. We tested the importance of calcium voltage-gated channels (VOCs) and thus the electrical communication, by using different inhibitors of the VOCs. We also tested the importance of stretch-activated channels (SACs) by inhibiting contraction of individual SMCs. Finally, we verified the importance of gap junctions and thus of chemical and/or electrical coupling. We applied a gap junction inhibitor and we also performed microinjection of fluorescent dyes in single SMCs. Our results provide evidence that under PE the synchronization of the SMCs recruitment occurred electrically through gap junctions. Under KCl, every SMC was simultaneously depolarized and calcium entered the cell simultaneously through VOCs. This leads to a global [Ca2+]i increase. When we studied the recruitment and synchronization of the SMCs under PE, we sometimes observed intercellular calcium waves propagating between SMCs in a direction corresponding to the arterial axis. In chapter 5, we used a tracking process to quantify these intercellular waves. We found that intercellular calcium waves propagating along the strip were linked to the local contraction variations. The velocity of both waves are similar (~60 µm/s). This method of tracking is particularly powerful for the study of cellular communication in arteries. It has the advantage of allowing the correlation of calcium with contraction dynamics. The role of the endothelium for vasomotion is matter of debate. SMCs and ECs are electrically and chemically coupled allowing exchange of intercellular signaling. In chapter 6, we investigated heterocellular communication at the cellular level in order to understand how SMCs can influence ECs calcium dynamics. SMCs of the arterial strip were stimulated by KCl and by PE. We verified that PE and KCl did not directly act on the calcium concentration of ECs. Depending on vasoconstrictor concentration, calcium concentration increased in some ECs 5 to 11 s after that a calcium concentration increase was observed in SMCs. The existence of this time interval suggests a chemical coupling between SMCs and ECs through gap junctions. Most probable chemical mediators are either calcium or inositol 1,4,5-trisphosphate (IP3). To discriminate between calcium and IP3 diffusion, first a phospholipase C inhibitor was applied to prevent IP3 production in response to the calcium concentration increase in SMCs. Under this inhibitor and KCl, all SMCs presented a global and synchronous calcium concentration increase, but no calcium concentration variations in ECs were detected. Secondly, 2-Aminoethoxydiphenylborate, an inhibitor of IP3-induced calcium release, reduced the number of flashing ECs by 75±3 %. The number of flashing ECs was also significantly reduced when palmitoleic acid, a gap junction uncoupler was added. Our results were compatible with a heterocellular communication based on IP3 diffusion through gap junctions from SMCs to ECs. The last chapter summarizes our findings and provides our conclusions with some perspectives.