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.     

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.     

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.     

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.     

Gap junctional communication and compaction during preimplantation stages of mouse development

The ability of gap junction antibodies to block dye transfer and electrical coupling was examined in the compacted 8-cell mouse zygote. In control zygotes, Lucifer yellow injected into 1 cell transferred to the rest of the embryo. When antibodies raised against the major protein extracted from gap junctions were co-injected with Lucifer yellow, dye transfer failed in 86% of the zygotes tested and electrical coupling was almost completely inhibited. Subsequently, the antibody-containing cells were extruded. When the antibodies were injected into 1 cell at the 2-cell stage, 82% of the zygotes divided normally to the 8-cell stage. Cells containing gap junction antibodies were uncompacted, but continued to divide. We conclude that these antibodies inhibit gap junctional communication in the early mouse zygote and that communication through gap junctions may be involved in the maintenance of compaction.     

On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells

Pancreatic beta-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in beta-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.     

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     

Changes in intercellular exchange intensity in an L cell culture]

Intercellular junctions permeable to fluorescein Na were studied with the aid of intracellular glass microelectrodes in the cultures of transformed mouse-embryo cells (L-strain). The degree of coupling was correlated with the cell culture density. In addition, the degree of coupling increased after short-time incubated of the cells with 1mM lanthanum. Such an incubation did not influence other features of cells: the value of the membrane potential, the character of growth or the inclusion of the vital dye neutral red. Some other factors stimulated cellular aggregation (concanavalin A, protamine, decreasing the pH) but did not influence the degree of coupling.     

Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca(2+)](i)) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca(2+)](i) waves. N(G)-nitro-l-arginine methyl ester (10 microM) did not mimic this effect, and dibutyryl cGMP (300 muM) failed to resynchronize [Ca(2+)](i) waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, (37,43)Gap27 and (40)Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca(2+)](i) waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling

Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.     

The third pathway: endothelium-dependent hyperpolarization.

In response to various neurohumoral substances endothelial cells release nitric oxide (NO), prostacyclin and produce hyperpolarization of the underlying vascular smooth muscle cells, possibly by releasing another factor termed endothelium-derived hyperpolarizing factor (EDHF). EDHF-mediated responses are sensitive to the combination of two toxins, charybdotoxin plus apamin, but do not involve ATP-sensitive or large conductance calcium-activated potassium channels. As hyperpolarization of the endothelial cells is required in order to observe endothelium-dependent hyperpolarization, and electrical coupling through myo-endothelial gap junctions may explain the phenomenon. An alternative explanation is that the hyperpolarization of the endothelial cells causes an efflux of potassium that in turn activates the inwardly rectifying potassium conductance and the Na+/K+ pump of the smooth muscle cells. Endothelial cells produce metabolites of the cytochrome P450-monooxygenase that activate BKCa, and induce hyperpolarization of coronary arterial smooth muscle cells. The elucidation of the mechanism underlying endothelium-dependent hyperpolarization and the discovery of specific inhibitors of the phenomenon are prerequisite for the understanding of the physiological role of this alternative endothelial pathway involved in the control of vascular tone in health and disease.     

Rhythmic coupling among cells in the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells. There is also evidence that the cells within the SCN are coupled to one another and that this coupling is important for the normal functioning of the circadian system. One mechanism that mediates coordinated electrical activity is direct electrical connections between cells formed by gap junctions. In the present study, we used a brain slice preparation to show that developing SCN cells are dye coupled. Dye coupling was observed in both the ventrolateral and dorsomedial subdivisions of the SCN and was blocked by application of a gap junction inhibitor, halothane. Dye coupling in the SCN appears to be regulated by activity-dependent mechanisms as both tetrodotoxin and the GABA(A) agonist muscimol inhibited the extent of coupling. Furthermore, acute hyperpolarization of the membrane potential of the original biocytin-filled neuron decreased the extent of coupling. SCN cells were extensively dye coupled during the day when the cells exhibit synchronous neural activity but were minimally dye coupled during the night when the cells are electrically silent. Immunocytochemical analysis provides evidence that a gap-junction-forming protein, connexin32, is expressed in the SCN of postnatal animals. Together the results are consistent with a model in which gap junctions provide a means to couple SCN neurons on a circadian basis.     

Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.     

Varied effects of 1-octanol on gap junctional communication between ovarian epithelial cells and oocytes of Oncopeltus fasciatus, Hyalophora cecropia, and Drosophila melanogaster

In insect gap junctions, species-specific differences occur in response to the purported gap junction uncoupling agent, 1-octanol. Changes in gap junctional communication between oocytes and their epithelial cells following treatment with 1-octanol were assayed in Oncopeltus fasciatus (the milkweed bug), Hyalophora cecropia (the American silk moth), and Drosophila melanogaster. In all three species, microinjection of untreated control follicles with Lucifer yellow CH revealed extensive dye coupling among epithelial cells and between epithelial cells and their oocytes. Also for all three species, treatment with octanol appeared to completely block dye coupling and increase oocyte input resistance. The effect on electrical coupling varied. In Drosophila, octanol diminished the electrical coupling from 64% (0.64 coupling coefficient) in controls to 53% in treated follicles. In Hyalophora, the coupling ratio remained the same following treatment. In Oncopeltus, octanol actually increased the electrical coupling ratio from 84% in controls to 94% in treated follicles. While 0.5 mM octanol left some Oncopeltus epithelial cells dye coupled to the oocyte, the electrical coupling ratio was increased slightly more by this concentration than by 1 or 5 mM octanol solutions, although the differences were not significant. While input resistance (R(o )) increased in all three following treatment with octanol, there was considerable difference in the magnitude of the response. Average oocyte R(o ) for Oncopeltus increased the least of the three species, rising from 196-240 kOhm. Both Hyalophora, with a nearly fourfold increase from 230-900 kOhm or more, and Drosophila, with a twofold increase from 701 kOhm to over 1.2 MegOhm showed much larger changes. Results shown here indicate that insect gap junctions have more varied responses to this common gap junction antagonist than have been reported for their vertebrate counterparts. Arch.     

Electrical properties of developing oocytes of the migratory locust, Locusta migratoria.

The electrical properties of developing nonfertilized oocytes of Locusta migratoria were studied, using intracellular microelectrodes. The inseries potential of the combined oomembrane and of the follicular cells was about 20 mV in the youngest oocytes. It increased as the oocytes developed and it reached a plateau of about 50 mV before full maturation, generally four to seven oocytes away from the fully-developed terminal oocyte. Current-voltage relations were always linear for hyperpolarizing currents. Most oocytes exhibited, however, rectification to outward current. Input resistance values varied with oocyte size from about 5 X 10(6) ohm for young oocytes to about 0.2 X 10(6) ohm for the more developed ones. Some oocytes displayed a transient depolarization on turning off a hyperpolarizing step of current. This depolarization was not correlated with the size of the oocyte or with any observed morphological feature. Any two adjacent oocytes were electrotonically coupled. A single ovariole thus represented a longitudinal chain of developing oocytes which were connected electrically. This was supported by electron microscope observations which revealed junctions partially impermeable to lanthanum and gap junctions between the follicular cells themselves and between follicular cells and oocytes. The coupling coefficient was dependent on the direction of current flow. The attenuation of voltage along an ovariole was always greater at the distal than at the proximal side.     

Cell to cell communication and pH in the frog lens

Fiber cells of the lens are electrically and diffusionally interconnected through extensive gap junctions. These junctions allow fluxes of small solutes to move between inner cells and peripheral cells, where the majority of transmembrane transport takes place. We describe here a method utilizing two intracellular microelectrodes to measure the cell to cell resistance between fiber cells at any given distance into the intact lens. We also use ion-sensitive microelectrodes to record intracellular pH at various depths in the intact lens. We find that gap junctions connecting inner fiber cells differ in pH sensitivity as well as normal coupling resistance from those connecting peripheral cells. The transition occurs in a zone between 500 and 650 microns into the lens. Fiber cells peripheral to this zone have a specific coupling resistance of 1.1 omega cm2, whereas those inside have a specific coupling resistance of 2.7 omega cm2. However, when the cytoplasm of fiber cells is acidified by bubbling with CO2, peripheral cells uncouple and the cell to cell resistance goes up more than 40-fold, whereas junctions inside this zone are essentially unaffected by changes in intracellular pH. In a normal frog lens, the intracellular pH in fiber cells near the lens surface is 7.02, a value significantly alkaline to electrochemical equilibrium. Our data suggest that Na/H exchange and perhaps other Na gradient-dependent mechanisms in the peripheral cells maintain this transmembrane gradient. Deep in the lens, the fiber cell cytoplasm is significantly more acidic (pHi 6.81) due to influx of hydrogen across the inner fiber cell membranes and production of H+ by the inner fiber cells. Because of the normally acid cytoplasm of interior fiber cells, their loss of gap junctional sensitivity to pH may be essential to lens survival.     

An essential role for connexin43 gap junctions in mouse coronary artery development

Connexin43 knockout mice die neonatally from conotruncal heart malformation and outflow obstruction. Previous studies have indicated the involvement of neural crest perturbations in these cardiac anomalies. We provide evidence for the involvement of another extracardiac cell population, the proepicardial cells. These cells give rise to the vascular smooth muscle cells of the coronary arteries and cardiac fibroblasts in the heart. We have observed the abnormal presence of fibroblast and vascular smooth muscle cells in the infundibular pouches of the connexin43 knockout mouse heart. In addition, the connexin43 knockout mice exhibit a variety of coronary artery patterning defects previously described for neural crest-ablated chick embryos, such as anomalous origin of the coronary arteries, absent left or right coronary artery, and accessory coronary arteries. However, we show that proepicardial cells also express connexin43 gap junctions abundantly. The proepicardial cells are functionally well coupled, and this coupling is significantly reduced with the loss of connexin43 function. Further analysis revealed an elevation in the speed of cell locomotion and cell proliferation rate in the connexin43-deficient proepicardial cells. A parallel analysis of proepicardial cells in transgenic mice with dominant negative inhibition of connexin43 targeted only to neural crest cells showed none of these coupling, proliferation or migration changes. These mice exhibit outflow obstruction, but no infundibular pouches. Together these findings indicate an important role for connexin43 in coronary artery patterning, a role that probably involves the proepicardial and cardiac neural crest cells. We discuss the potential involvement of connexin43 in human cardiovascular anomalies involving the coronary arteries.     

Nitric oxide specifically reduces the permeability of Cx37-containing gap junctions to small molecules

Gap junction intercellular communication (GJIC) plays a significant role in the vascular system. Regulation of GJIC is a dynamic process, with alterations in connexin (Cx) protein expression and post-translational modification as contributing mechanisms. We hypothesized that the endothelial autacoid nitric oxide (NO) would reduce dye coupling in human umbilical vein endothelial cells (HUVECs). In our subsequent experiments, we sought to isolate the specific Cx isoform(s) targeted by NO or NO-activated signaling pathways. Since HUVEC cells variably express three Cx (Cx37, Cx40, and Cx43), this latter aim required the use of transfected HeLa cells (HeLaCx37, HeLaCx43), which do not express Cx proteins in their wild type form. Dye coupling was measured by injecting fluorescent dye (e.g., Alexa Fluor 488) into a single cell and determining the number of stained adjacent cells. Application of the NO donor SNAP (2 microM, 20 min) reduced dye coupling in HUVEC by 30%. In HeLa cells, SNAP did not reduce dye transfer of cells expressing Cx43, but decreased the dye transfer from Cx37-expressing cells to Cx43-expressing cells by 76%. The effect of SNAP on dye coupling was not mediated via cGMP. In contrast to its effect on dye coupling, SNAP had no effect on electrical coupling, measured by a double patch clamp in whole cell mode. Our results demonstrate that NO inhibits the intercellular transfer of small molecules by a specific influence on Cx37, suggesting a potential role of NO in controlling certain aspects of vascular GJIC.     

Structural and functional studies of myometrial gap junctions

Gap junctions are believed to be sites of metabolic and electrical coupling between cells. These contacts are present between myometrial cells immediately prior to and during parturition. We report the results of studies to investigate the control and the function of myometrial gap junctions. Injection of estradiol (500 micrograms/day) with or without progesterone into immature and ovariectomized mature rats demonstrated that estradiol stimulated whereas progesterone suppressed gap junction formation. Indomethacin treatment was also shown to potentiate the action of estradiol. Also, pregnant rats treated with oestradiol developed numerous myometrial gap junctions and aborted their fetuses. These results suggest that the steroid hormones and prostaglandins may control myometrial gap junction development. Diffusion studies of 3H-2-deoxyglucose in longitudinal myometrial strips revealed a significant increase in the diffusion coefficient in delivering versus ante-partum rat tissues. This indicates that there is increased metabolic transfer during parturition when gap junctions are present. The results of these studies show that steroid hormones and prostaglandins may regulate myometrial gap junctions and that metabolic, as well as electrical coupling, of uterine smooth muscle cells increase at parturition concomitant with the development of gap junctions.