Dye and electrical coupling of endothelial cells in situ

Electron microscopic studies show that endothelial cells of pig coronary arteries are linked by gap junctions. We investigated the dye and electrical coupling of these junctions in a strip of pig coronary artery in vitro. The membrane potential of two neighbouring (about 0.2 mm) endothelial cells were simultaneously recorded with two microelectrodes. The fluorescent dye lucifer yellow was microiontophoretically injected through one of the microelectrodes. The endothelial cells in situ were dye and electrically coupled. The dye coupling extended parallel to the longitudinal axis of the arteries. We conclude that an electrical message like the bradykinin and substance P hyperpolarizations of the endothelial cells can be conveyed electrotonically by the endothelium along the longitudinal axis of arteries.     

Lucifer yellow - an angel rather than the devil

The fluorescent dye Lucifer yellow (LY) was introduced in 1978, and has been extremely useful in studying cell structure and communications. This dye has been used mostly for labelling cells by intracellular injection from microelectrodes. This review describes the numerous applications of LY, with emphasis on the enteric nervous system and interstitial cells of Cajal. Of particular importance is the dye coupling method, which enables the detection of cell coupling by gap junctions.     

Physiological and morphological evidence for coupling in mouse salivary gland acinar cells

Three experimental techniques were employed to examine coupling between acinar cells of the mouse salivary gland. Passage of DC current pulses via intracellular microelectrodes between neighboring cells showed that small ions could be directly passed from one cell to another. Intracellular iontophoresis of the dye Lucifer Yellow CH into a single cell indicated that small molecules could spread by means of intercellular cytoplasmic bridges througout an acinus and, occasionally, into cells of adjacent acini. Freeze-fracture replicas of acinar cell membranes indicated the presence of gap junctions which were correlated with both electrical and dye coupling experiments. Suggestions are made for the function of direct intercellular exchange in salivary secretory cells. The role of electrical coupling in coordination of the activity of different secretory cell types is discussed as one possible function.     

Longitudinal body wall muscles are electrically coupled across the segmental boundary in the third instar larva ofDrosophila melanogaster

Longitudinal body wall muscles in the third instar larva of the fruitfly,Drosophila melanogaster, were systematically examined for electrical and dye coupling. These muscle cells were found to be electrically coupled but rarely dye-coupled across the segmental boundary. The inter-segmental coupling coefficients between muscle #6s and muscle #7s across the segmental boundary were 0.33 ± 0.09 (mean ± S.D.,n = 12) and 0.43 ± 0.09 (n = 5), respectively, which are much larger than values previously reported inDrosophila but similar to those reported in the blowfly and hawkmoth. By contrast, the intra-segmental coupling coefficient between muscles #6 and #7 was smaller, 0.16 ± 0.08 (n = 28). Other muscle cells which had apparent physical contacts with these longitudinal muscles were examined but were not electrically coupled to them. Nerve-evoked as well as miniature excitatory junctional potentials were found also electrotonically spread across the segmental boundary. The inter-segmental coupling between muscle #6s was not blocked by the gap junction inhibitors halothane or 1-octanol. Functional significance of this electrical coupling is apparently in coordination of larval body movements.     

On the dynamics of electrically-coupled neurons with inhibitory synapses

We study the dynamics and bifurcations of noise-free neurons coupled by gap junctions and inhibitory synapses, using both delayed delta functions and alpha functions to model the latter. We focus on the case of two cells, as in the studies of Chow and Kopell (2000) and Lewis and Rinzel (2003), but also show that stable asynchronous splay states exist for globally coupled networks of N cells dominated by subthreshold electrical coupling. Our results agree with those of Lewis and Rinzel (2003) in the weak coupling range, but our Poincaré map analysis yields more information about global behavior and domains of attraction, and we show that the explicit discontinuous maps derived using delayed delta functions compare well with the continuous history-dependent, implicitly-defined maps derived from alpha functions. We find that increased bias currents, super-threshold electrical coupling and synaptic delays promote synchrony, while sub-threshold electrical coupling and fast synapses promote asynchrony. We compare our analytical results with simulations of an ionic current model of spiking cells, and briefly discuss implications for stimulus response modes of locus coeruleus and for central pattern generators. Action Editor: F. Skinner     

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

The network of interstitial cells of Cajal associated with Auerbach’s (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach’s plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach’s plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities. Received: 21 July 1995 / Accepted: 22 April 1998     

Opposing Effects of Nitric Oxide on Different Connexins Expressed in the Vascular System

Gap junctions—clusters of intercellular channels built by connexins (Cx)—are thought to be important for vascular cell functions such as differentiation, control of tone, or growth. In the vascular system, gap junctions can be formed by four different connexins (Cx37, Cx40, Cx43 and Cx45). The permeability of these connexin-formed gap junctions determines the amount of intercellular coupling and can be modulated by several vasoactive substances such as prostacyclin or nitric oxide (NO). We demonstrate here that NO has specific effects on certain connexins. Using two different techniques—injection of a fluorescent dye in single cells as well as detection of the de novoformation of gap junctions by a flow cytometry based technique—we found that NO decreases the functional coupling in Cx37 containing gap junctions whereas it increases the de novoformation of gap junctions containing Cx40. We conclude that NO, in addition to its known vasomotor effects, has a novel role in controlling intercellular coupling resulting in opposing effects depending on the specific connexin expressed in the cells.     

Phytochrome control of electrical potentials and intercellular coupling in oat-coleoptile tissue

Summary Microelectrodes were used to demonstrate two electrical responses which occur in oat (Avena sativa L.) coleoptile parenchyma-cells during exposure to red light. The membrane potential of these cells depolarized 5–10 mV in several seconds in red light and repolarized more slowly in far-red light. By pulsing current through the cells, it was found that cellular coupling along the longitudinal axis of the coleoptile increased about 2-fold in red light, but that coupling along the lateral axis was not affected. The rapid changes in membrane potential are consistent with the idea of a membrane locale for early phytochrome action. The coupling experiments suggest that phytochrome may also affect plasmodesmata in this system.     

A coronary artery disease-associated gene product, JCAD/KIAA1462, is a novel component of endothelial cell-cell junctions

Cell–cell junctions play crucial roles in the organization and function of epithelial and endothelial cellular sheets. Here, we have identified the protein product for KIAA1462 gene, whose single nucleotide polymorphisms (SNPs) have recently reported to be associated with coronary artery disease, as a novel component of cell–cell junctions. We propose the name of KIAA1462 protein junctional protein associated with coronary artery disease (JCAD). JCAD is a ∼145 kDa protein without any known domains but contains a proline-rich region. Immunolocalization studies revealed that JCAD is specifically localized at cell–cell junctions in endothelial cells but not in epithelial cells. The accumulation of JCAD at cell–cell junctions in cultured endothelial cells was impaired by RNAi-mediated suppression of VE-cadherin expression. In cell adhesion-deficient mouse L fibroblasts, JCAD was recruited to cell–cell contacts when cadherin-mediated cell–cell adhesion was induced. These results indicate that JCAD is a component of VE-cadherin-based cell–cell junctions in endothelial cells. This study also suggests the implication of endothelial cell–cell adhesion in coronary artery disease.     

ELECTROPHYSIOLOGICAL INVESTIGATIONS OF THE RED ALGA GRIFFITHSIA PAGIFIG AKYL. 1

The membrane potentials of the plasmalemma and tonoplast in Griffithsia pacifica Kyl were measured and shown to be significantly different. No consistent cell wall potential was discovered. Adjacent cells in the filament were demonstrated to be electrolonically coupled with a mean coupling coefficient of 0.13. Cobaltions did not move between cells. No dye coupling was seen using the fluorescent dye Lucifer yellow CH. These observations provide new information on the electrophysiology of red algae and indicate that there is intercellular continuity between the cells of a filament of Griffithsia pacifica.     

Communication compartments in the ectoderm of embryos of Patella vulgata

Summary Patterns of gap junctional communication in the ectoderm of embryos of Patella vulgata have been studied by intracellular injection of the fluorescent dye Lucifer Yellow, and by analysis of its subsequent spread to adjacent cells (dye-coupling). We found that dye-coupling became progressively restricted to different domains of the ectoderm, forming communication compartments. These communication compartments are characterized by their high coupling abilities within the compartment, and reduction of coupling across their boundaries. During development, the pretrochal (anterior) ectoderm becomes subdivided into two communication compartments, the apical organ and the anlage of the head ectoderm. The posttrochal (posterior) ectoderm becomes subdivided into different communication compartments in two successive phases. Firstly, in the 15-h embryo the dorsal and ventral domains of the ectoderm form separate communication compartments. A dorso-ventral communication boundary restricts the passage of dye between the two domains. Secondly, in the 24-h embryo dye-coupling becomes further compartmentalized in both the dorsal and ventral domains. These compartments correspond to the anlagen of different ectodermal structures. In order to study whether any level of coupling persists between the ectodermal compartments we injected currents through a microelectrode inserted into one cell of one compartment and monitored its spread by means of a second microelectrode inserted into one cell of another compartment (electrical coupling). Despite the absence of dye-coupling, electrical coupling between the ectodermal dye-coupling compartments was detected, which suggests that some level of communication is maintained between compartments. Our results demonstrate that within the ectoderm layer of Patella vulgata the transfer of dyes becomes progressively restricted to communication compartments and, concomitantly with the specification of the different ectodermal anlagen, these compartments become subdivided into smaller communication compartments.     

Local nonuniformities in the horizontal cell syncytium of the turtle retina

Electrical coupling between horizontal cells of the turtle retina was investigated by means of two microelectrodes, stimulating and recording, inserted into neighboring cells at a fixed horizontal distance apart. Morphological coupling was estimated by studying the flow of the luminescent dye Lucifer yellow. The presence of electrical coupling was confirmed between structures of the same type (L₁ with axon terminals, L₂ withcell bodies, R/G with cells of color type) and absence of coupling between cells of different types was confirmed, although L₁ and L₂ are connected with each other directly by thin axons. The degree of electrical coupling in the syncytium of axon terminals (L₁), with a short (50 µ or less) but fixed distance between the electrodes, may vary by several times depending on the position of the microelectrodes. This local nonuniformity of coupling can be explained by the structural nonuniformity of the network of interconnected axon terminals. Local structural nonuniformities can hardly affect the functional properties of the horizontal cell syncytium under the conditions of photic stimulation of the retina.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 239–245, March–April, 1985.     

Junctional transfer in cultured vascular endothelium: II. Dye and nucleotide transfer

Summary Vascular endothelial cultures, derived from large vessels, retain many of the characteristics of theirin vivo counterparts. However, the observed reduction in size and complexity of intercellular gap and tight junctions in these cultured cells (Larson, D.M., and Sheridan, J.D., 1982,J. Cell Biol. 92:183) suggests that important functions, thought to be mediated by these structures, may be alteredin vitro. In our continuing studies on intercellular communication in vessel wall cells, we have quantitated the extent of junctional transfer of small molecular tracers (the fluorescent dye Lucifer Yellow CH and tritiated uridine nucleotides) in confluent cultures of calf aortic (BAEC) and umbilical vein (BVEC) endothelium. Both BAEC and BVEC show extensive (and quantitatively equivalent) dye and nucleotide transfer. As an analogue of intimal endothelium, we have also tested dye transfer in freshly isolated sheets of endothelium. Transfer in BAEC and BVEC sheets was more rapid, extensive and homogeneous than in the cultured cells, implying a reduction in molecular coupling as endothelium adapts to culture conditions. In addition, we have documented heterocellular nucleotide transfer between cultured endothelium and vascular smooth muscle cells, of particular interest considering the prevalence of myo-endothelial junctionsin vivo. These data yield further information on junctional transfer in cultured vascular endothelium and have broad implications for the functional integration of the vessel wall in the physiology and pathophysiology of the vasculature.     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

Intercellular coupling among interstitial cells of Cajal in the guinea pig small intestine

A major difficulty in the investigation of interstitial cells of Cajal (ICC) is in identifying these cells within intact, living gastrointestinal tissues. To overcome this difficulty we developed a method to visualize ICC in the myenteric plexus region (ICC-MP) of the guinea pig ileum. Cells were identified with Nomarski optics and were injected with the fluorescent dye Lucifer yellow. The identity of the cells as ICC was verified by immunohistochemical labeling for the protein c-Kit. Using the dye coupling method we found that 24.4% (93/381) of ICC-MP were coupled to 1-21 other ICC. Octanol reduced dye coupling incidence among ICC-MP to 2% (1/49). Raising the pH in the medium to 7.8-7.9 increased the dye-coupling incidence to 86% (37/43, P<0.001). Lowering the pH to 6.4-6.8 had the opposite effect (coupling incidence 1/44). These findings demonstrate that ICC are mutually connected by channels, apparently gap junctions, that can allow the passage of both electrical currents and small molecules. As it was modulated by pH, it is likely that ICC coupling is under physiological control.     

Ascorbate uptake in pig coronary artery endothelial cells

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When ¹⁴C-ascorbate uptake in endothelial cells was examined as ¹⁴C or by HPLC, the two values did not differ from each other. ¹⁴C-ascorbate uptake was Na⁺-dependent, stereoselective for l-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: K_m = 27± 3 M (Hill coefficient = 1) for ascorbate and K_m = 73± 14 mM (Hill coefficient = 2) for Na⁺. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect. (Mol Cell Biochem 271: 43–49, 2005)     

Electrical coupling between cells in islets of langerhans from mouse

Summary Two microelectrodes have been used to measure membrane potentials simultaneously in pairs of mouse pancreatic islet cells. In the presence of glucose at concentrations between 5.6 and 22.2mm, injection of currenti into cell 1 caused a membrane potential change in this cell,V ₁, and, provided the second microelectrode was less than 35 m away, in a second impaled cell 2,V ₂. This result establishes that there is electrical coupling between islet cells and suggests that the space constant of the coupling ratio within the islet tissue is of the order of a few -cell diameters. The current-membrane potential curvesi-V ₁ andi-V ₂ are very similar. By exchange of the roles of the microelectrodes, no evidence of rectification of the current through the intercellular pathways was found. Removal of glucose caused a rapid decrease in the coupling ratioV ₂ /V ₁ . In steady-state conditions, the coupling ratio increases with the concentration of glucose in the range from 0 up to 22mm. Values of the equivalent resistance of the junctional and nonjunctional membranes have been estimated and found to change with the concentration of glucose. Externally applied mitochondrial blockers induced a moderate increase in the junctional resistance possibly mediated by an increase in intracellular Ca²⁺.     

Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB1

Recent studies have indicated that the inflammasome plays a critical role in the pathogenesis of vascular diseases. However, the pathological relevance of this inflammasome activation, particularly in vascular cells, remains largely unknown. Here, we investigated the role of endothelial (Nucleotide‐binding Oligomerization Domain) NOD‐like receptor family pyrin domain containing three (Nlrp3) inflammasomes in modulating inter‐endothelial junction proteins, which are associated with endothelial barrier dysfunction, an early onset of obesity‐associated endothelial injury. Our findings demonstrate that the activation of Nlrp3 inflammasome by visfatin markedly decreased the expression of inter‐endothelial junction proteins including tight junction proteins ZO‐1, ZO‐2 and occludin, and adherens junction protein VE‐cadherin in cultured mouse vascular endothelial (VE) cell monolayers. Such visfatin‐induced down‐regulation of junction proteins in endothelial cells was attributed to high mobility group box protein 1 (HMGB1) release derived from endothelial inflammasome‐dependent caspase‐1 activity. Similarly, in the coronary arteries of wild‐type mice, high‐fat diet (HFD) treatment caused a down‐regulation of inter‐endothelial junction proteins ZO‐1, ZO‐2, occludin and VE‐cadherin, which was accompanied with enhanced inflammasome activation and HMGB1 expression in the endothelium as well as transmigration of CD43⁺ T cells into the coronary arterial wall. In contrast, all these HFD‐induced alterations in coronary arteries were prevented in mice with Nlrp3 gene deletion. Taken together, these data strongly suggest that the activation of endothelial Nlrp3 inflammasomes as a result of the increased actions of injurious adipokines such as visfatin produces HMGB1, which act in paracrine or autocrine fashion to disrupt inter‐endothelial junctions and increase paracellular permeability of the endothelium contributing to the early onset of endothelial injury during metabolic disorders such as obesity or high‐fat/cholesterol diet.     

Responses of neuroblastoma cells to iontophoretically applied acetylcholine

Dissociated mouse neuroblastoma cells were studied in vitro by using intracellular microelectrodes for electrical stimulation and recording. Some, but not all cells, which exhibited well developed action potentials to electrical stimulation also showed changes in membrane potential to iontophoretically applied acetylcholine (ACh). The types of responses to ACh varied. Short latency depolarizing responses to pulses of ACh (similar to those obtained with skeletal muscle) as well as sustained depolarization to steady ACh application (D response) occurred. A longer latency prolonged hyperpolarizing response (H response) and bi- and triphasic combinations of H and D responses were also seen. Pairs of cells showing morphologic contact were tested for the occurrence of effective synaptic coupling by placing intracellular microelectrodes in each cell. In none of 95 cases tested did spike activity produced by direct electrical stimulation of one cell elicit a synaptic potential of 200 μv or more in the other.