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

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.

Calcium Dynamics and Intercellular Communication in Arterial Smooth Muscle Cells in Vitro

Nadia Halidi

Tissue blood flow is controlled by changes in the diameter of the arteries and arterioles through coordinated contraction and relaxation of smooth muscle cells (SMCs) within the vascular wall. The contraction of SMCs is primarily regulated by the intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i, in response to stimuli, can propagate from cell to cell, as an intercellular Ca2+ wave along the vessel wall and can activate the process of contraction. The aim of this thesis is to elucidate the mechanisms underlying intercellular Ca2+ wave propagation between SMCs. In the first part of this thesis, we used A7r5 cells, a rat aortic SMC line, loaded with the fluorescent Ca2+ dye Fluo-4 to study intercellular Ca2+ wave propagation. Local mechanical stimulation evoked a Ca2+ wave in the stimulated cell that failed to propagate to neighboring cells. Using primary cultured rat mesenteric smooth muscle cells (pSMCs) instead, intercellular Ca2+ wave propagation was observed. To understand the difference in junctional communication between A7r5 and pSMCs, we investigated the expression of connexin37 (Cx37), Cx40, Cx43 and Cx45. RNA and protein analysis demonstrated that Cx40 – in contrast to A7r5 cells – is not expressed in pSMCs. To confirm that coexpression of Cx40 and Cx43 interfered with junctional communication, we used 6B5N cells, a clone of A7r5 cells with a higher Cx43:Cx40 expression ratio. Junctional communication, assessed by transfer of Lucifer Yellow and propagation of Ca2+ waves, was comparable between 6B5N cells and pSMCs. In addition, Ca2+ wave propagation was inhibited with the connexin-mimetic peptide 43Gap 26, that targets Cx43. Our results demonstrate that Cx43 gap junctions are primarily involved in mediating intercellular Ca2+ waves between pSMCs, and the coexpression of Cx43 with Cx40 may interfere with Cx43 gap junction formation, affecting cell-cell communication. In the second part of this thesis, we applied the microcontact printing technique to culture pSMCs on collagen lines. The aligned arrangement of the cells facilitates the observation of Ca2+ wave progression from one cell to another. To induce a Ca2+ wave, a single pSMC was locally stimulated with a micropipette (transientmechanical stimulation) or by microejection of KCl. Mechanical stimulation evoked two distinct Ca2+ waves: 1) a fast wave (∼2 mm/s) that propagated to all observed neighbouring cells, and 2) a slow wave (∼20 µm/s), that was most often limited in propagation to the first cell. KCl induced only fast Ca2+ waves of the same velocity as the mechanically-induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors, showed that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca2+ influx, whereas, the slow wave was due to Ca2+ release primarily through IP3 receptors. Together, these results suggest a mechanism by which intercellular Ca2+ waves can propagate between SMCs of the arterial wall.

EPFL2011

Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro

Jean-Louis Bény, Jean-Jacques Meister, Alexander Schuster, Nikolaos Stergiopulos

The regulation of cytosolic-free calcium concentration of smooth-muscle and endothelial cells was mainly studied on cultured cells where the cross talk between these two coupled cell types is lost. In the present study, the cytosolic-free calcium concentration in the endothelial and the smooth-muscle cells was examined in an intact arterial wall in vitro. Strips of the main branch of rat mesenteric artery were used. Cytosolic-free calcium concentration [Ca2+]i was estimated by determining the fluorescence ratio of the two calcium probes, Fluo-4 and Fura red. The emitted fluorescence of both probes was measured with a confocal microscope. We showed that potassium and phenylephrine, which increase the cytosolic -free calcium concentration of the smooth-muscle cells, also indirectly influence the calcium concentration in the endothelial cells. By simultaneously determining [Ca2+]i in the endothelial and the smooth-muscle cells of an arterial strip, we observed that when calcium increases in the endothelial cells in response to acetylcholine, it slightly decreases in the smooth-muscle cells. We conclude that the regulation of [Ca2+]i in the arterial endothelial cell, depends according to the stimuli either upon the endothelial cells themselves, or upon the coupled smooth-muscle cells.

Elsevier2001

Activation and Spatial Organization of the Adenosine A2A Receptor in Supported Plasma Membrane Sheets

Sophie Roizard

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. The main mechanisms involving GPCR activation and signal transduction to the cell interior are understood but many of the processes which fine-tune GPCRs signalling need to be unraveled. The work performed in this thesis intended to develop new methods to study GPCRs in their native environment but in a simplified system compared to live cells. In this context, the tethering GPCR-containing membrane fragments on solid surfaces appeared to be interesting: it enabled studying the receptors in their real native environment with several high resolution imaging techniques and with the possibility to engineer and control the downstream intracellular signaling components. Two procedures have been established to produce plasma membrane supported on micrometer-sized porous beads or on microscope grids for fluorescence or electron microscopy studies. They have been applied in this thesis to study the Adenosine A2A receeptor (A2A) as a prototypical GPCR. The first procedure enabled the transfer of native membrane patches from live cells with an inside-out orientation onto lectin-coated beads. Using heterologously expressed A2ARs carrying a yellow fluorescent protein (A2AR-Citrine) we showed that the tethered membranes were simultaneously accessible from both sides and comprised fully functional receptors in terms of ligand and G protein binding. These beads were investigated by confocal (Chapter 2) and single molecule (Chapter 3) fluorescence microscopies to study agonist and antagonist binding to the A2AR and the assembly and disassembly of the ternary complexes agonist-A2AR-Gαβγ proteins. The interactions between the different signalling partners could be observed in real time using multicolor fluorescence microscopy. These beads were also used in collaboration with colleagues for screening ligand binding to other transmembrane receptors. The second procedure produced GPCRs-containing plasma membrane fragments ripped-off on electron microscope grids for investigation by transmission electron microscopy (TEM) (Chapter 4). This procedure was improved from an existing protocol in order to produce reproducibly and with a high yield large membrane sheets and to preserve the membrane-associated cytoskeleton during the ripping-off. A2AR-Citrine were specifically gold immuno-labelled on their extracellular terminus using the FLAG-tag or their intracellular side by targeting the protein Citrine. The high resolution of TEM showed that the gold immuno-labelled A2AR-Citrine clustered and compartmentalized on actin-based cytoskeletton. This approach provided the ability to sample multiple single cells on a single grid and presented excellent potential to probe and unravel the organization of protein signalling networks in direct association with the plasma membrane-associated cytoskeleton.