An electron-microscopic study of smooth muscle cell dye coupling in the pig coronary arteries. Role of gap junctions (original) (raw)
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Experientia, 1990
The dye Lucifer Yellow was injected into single smooth muscle cells in the guinea pig small intestine in order to study intercellular coupling. Dye-coupling was observed in both the circular and longitudinal muscle layers and was markedly reduced when the intercellular pH was lowered. These results suggest the presence of gap junctions among intestinal muscle cells, but are inconsistent with previous ultrastructural studies that failed to demonstrate such junctions in the longitudinal muscle.
The American journal of physiology
The distributions of connexin 43 (Cx43) and connexin 40 (Cx40) in smooth muscle and endothelium of resistance vessels were examined using indirect immunofluorescence techniques coupled with confocal microscopy. Cx43 and Cx40 were found in smooth muscle and endothelium. Similar staining patterns were found in microvessel samples from brain and cremaster of the rat and from arterioles of the hamster cheek pouch. Double-labeling studies showed a high degree of colocalization of Cx40 with Cx43, suggesting the presence of multiple connexins within a single junctional plaque. Quantitative comparisons were made of the fluorescent patterns in the endothelium and smooth muscle of rat brain arterioles. Cx43 and Cx40 plaque diameters were 0.9 +/- 0.1 and 0.8 +/- 0.1 (SE) microns, respectively, in the endothelial layer and 0.5 +/- 0.1 and 0.5 +/- 0.1 microns, respectively, in the smooth muscle. There was no difference between mean plaque diameters of Cx43 and Cx40 in endothelium or smooth muscl...
Intercellular communication in cultured human vascular smooth muscle cells
American journal of physiology. Cell physiology, 2001
Intercellular communication through gap junction channels plays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV, capacitance vessel). Northern and Western blots documented the presence of connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA in cultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority of records, in both vessels, single-channel activity was dominated by a main-state conductance of 120 pS, with subconducting events comprising less than 10% of the amplitude histograms. However, some records showed "atypical" unitary events that had a co...
Cell and Tissue Research, 2002
Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro.
The American journal of physiology, 1995
The distributions of connexin 43 (Cx43) and connexin 40 (Cx40) in smooth muscle and endothelium of resistance vessels were examined using indirect immunofluorescence techniques coupled with confocal microscopy. Cx43 and Cx40 were found in smooth muscle and endothelium. Similar staining patterns were found in microvessel samples from brain and cremaster of the rat and from arterioles of the hamster cheek pouch. Double-labeling studies showed a high degree of colocalization of Cx40 with Cx43, suggesting the presence of multiple connexins within a single junctional plaque. Quantitative comparisons were made of the fluorescent patterns in the endothelium and smooth muscle of rat brain arterioles. Cx43 and Cx40 plaque diameters were 0.9 +/- 0.1 and 0.8 +/- 0.1 (SE) microns, respectively, in the endothelial layer and 0.5 +/- 0.1 and 0.5 +/- 0.1 microns, respectively, in the smooth muscle. There was no difference between mean plaque diameters of Cx43 and Cx40 in endothelium or smooth muscl...
Cell Calcium, 2005
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+ ] i ) increased in a subpopulation of ECs 5-11 s after a [Ca 2+ ] i 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+ ] i increase in SMCs. In high K + solution, all SMCs presented global and synchronous [Ca 2+ ] i increase, but no [Ca 2+ ] i 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 .
Utilization of a class of florescent cyanine dyes (Cy3™) for the assay of gap junctional communication by the dye transfer method was examined. When comp ed with Lucifer Yellow (LY), a commonly used tracer, microinjected Cy3 dye was found to yield similar degrees of cell coupling. Blockade of the transfer of both tracers by 12-O-tetradecanoylphorbol-13 acetate (TPA), which is known to cause closure of communicating channels, confirmed gap junctional mediation of dye movement. The fixability of a microinjected amine derivative of Cy3 dye demonstrated its compatibility with immunostaining protocols involving fluorescein isothiocyanate (FITC)-conjugated reagents. These results together with the brilliant fluorescence of Cy3 dyes suggest the potential of Cy3 reagents as additional tools to study gap junction function. © 1995.
Smooth muscle cells and interstitial cells of blood vessels
Cell Calcium, 2004
A rise in intracellular ionised calcium concentration ([Ca 2+ ] i) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle-and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca 2+ sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca 2+ ] i upon myocyte excitation; (2) spontaneous Ca 2+ sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca 2+-dependent K + and Cl − channels sense the local changes in [Ca 2+ ] i during a calcium spark and thereby couple changes in [Ca 2+ ] i within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP 3 Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.