Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration (Ca(2+)) increased in a subpopulation of ECs 5-11s after a Ca(2+) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the Ca(2+) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous Ca(2+) increase, but no Ca(2+) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).

Calcium dynamics and intercellular communication in arterial smooth muscle cells

Mathieu Lamboley

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.

EPFL2005

Intercellular calcium dynamics in smooth muscle cells

Dominique Seppey

Muscular arteries are able to actively modify their diameter by modulating the tone of the smooth muscle cells located within the arterial wall. A small variation of lumen diameter has a large influence on blood flow as well as on arterial pressure. The cardiovascular system pressure is mainly regulated by the resistance arteries and arterioles. Most of these arteries show cyclic variations of their diameter. This phenomenon is known as vasomotion and is in no way linked to any other physiological cycle like heartbeat or respiratory cycles. This phenomenon can be observed as well in vivo as in vitro. The arterial diameter oscillations are attributed to the contractile activity of the smooth muscle cells which is directly linked to their intracellular calcium concentration. Although observed since years, the mechanisms underlying vasomotion remain not well understood. It is known that arterial vasomotion is modified by apparition of abnormalities in the contractile mechanisms of arteries linked to cardiovascular diseases like hypertension. A better understanding of this phenomenon could therefore contribute to better treatments or diagnosis of such diseases. This thesis is divided in two parts which have each the goal to clear up a definite aspect of arterial vasomotion. The experiments have been performed in vitro on resistance arteries: the rat mesenteric arteries. These arteries have been observed under confocal fluorescence microscope allowing to quantify the intracellular calcium concentration in smooth muscle cells. In the first part of this work, we investigate the role played by the endothelium on arterial vasomotion. So far, contradictory results have been related in the literature about the role of the endothelium in the onset and maintenance of vasomotion. Even the elementary question of knowing if vasomotion can arise without an intact endothelium is quite controversial. To understand how the endothelium may either abolish or promote vasomotion, we have stimulated rat mesenteric arterial strips with a vasoconstrictor: the phenylephrine. Our results show that the endothelium is not necessary for vasomotion. However, when the endothelium is removed the phenylephrine concentration needed to induce vasomotion is lower and the rhythmic contractions occur for a narrower range of phenylephrine concentrations. By selectively inhibiting the endothelium-derived relaxing products, we demonstrate that these substances may either induce or abolish vasomotion. On the one hand, when the strip is tonically contracted in a non-oscillating state, an endothelium-derived relaxation may induce vasomotion. On the other hand, if the strip displays vasomotion, a relaxation may induce a transition to a non-oscillating state with a small contraction. In conclusion, our findings clarify the role of the endothelium on vasomotion and reconcile the seemingly contradictory observations reported in the literature. In the second part of this thesis we are interested in a vasomotion property not studied until now which is the propagation of vasomotion along arterial segments. The aim of this study is first to characterize these contraction waves, then in a second part to define what are the mechanisms allowing this propagation. We have stimulated rat mesenteric arterial strips with phenylephrine to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves. A velocity of about 100 µm/s was measured for the two kinds of waves and this velocity was independent of the presence of the endothelium on the strip. To investigate the calcium wave propagation mechanisms, we have elaborated a method allowing a phenylephrine stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low phenylephrine concentration was added on the whole strip, local phenylephrine stimulations induced calcium waves spreading over a finite distance of almost 400 µm. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage operated calcium channels, we have shown that the intercellular calcium wave propagation depends on the propagation of the smooth muscle cells depolarization. Finally, the results allow to propose a model of the mechanisms underlying the propagation of intercellular calcium waves along arterial segments during vasomotion.

EPFL2009

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Jean-Louis Bény, Jean-Jacques Meister

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.

Elsevier2004