Propagation of Action Potentials and the Structure of the Nexus in Cardiac Muscle

The hypothesis that the nexus is a specialized structure allowing current flow between cell interiors is corroborated by concomitant structural changes of the nexus and changes of electrical coupling between cells due to soaking in solutions of abnormal tonicity. Fusiform frog atrial fibers are interconnected by nexuses. The nexuses, desmosomes, and regions of myofibrillar attachment of this muscle are not associated in a manner similar to intercalated discs of guinea pig cardiac muscle. Indeed, nexuses occur wherever cell membranes are closely apposed. Action potentials of frog atrial bundles detected extracellularly across a sucrose gap change from monophasic to diphasic when the gap is shunted by a resistor. This indicates that action potentials are transmitted across the gap when sufficient excitatory current is allowed to flow across the gap. When the sucrose solution in the gap is made hypertonic, propagation past the gap is blocked and the resistance between the cells in the gap increases. Electron micrographs demonstrate that the nexuses of frog atrium and guinea pig ventricle are ruptured by hypertonic solutions.     

A STUDY OF THE STRUCTURE AND DISTRIBUTION OF THE NEXUS

Nexuses, that is, fusions of plasma membranes of adjacent cells, are described in mammalian smooth and cardiac muscle, median giant axon of earthworm, frog skin, and rat submandibular gland. In smooth muscle they usually occur where a process from one cell either meets a process of, or projects into a neighboring cell. On the other hand, in mammalian heart muscle and in earthworm giant axon the nexuses occur along the intercalated disc and intercellular segmental septa, respectively. Their occurrence between these excitable cells is correlated with propagation of action potentials by an electrical rather than chemical mechanism. Since the nexuses may offer pathways for electric current between cell interiors, it seems possible that they constitute a link in the structural basis for electrical transmission in these systems. In epithelia, nexuses usually appear as part of a terminal bar complex. This is true in the rat salivary gland studied here. In the epidermis of frog skin, nexuses are less numerous between the basilar columnar cells than between the subjacent squamous cells. The nexuses which occur in epithelia in frog skin and rat salivary gland are distributed as though to provide seals against electrochemical backleaks and sites of chemical exchange between cell interiors.     

Nexuses between the smooth muscle cells of the guinea-pig ileum

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.     

Hypertrophic smooth muscle

The smooth muscle cells of the circular musculature of the guinea pig ileum are connected by gap junctions (nexuses) which occupy about 0.21% of the cell surface. When the muscle hypertrophies in the portions of the ileum oral to an experimental stenosis, the muscle cells increase in size and number. Gap junctions become markedly larger than in control muscles and occupy 0.49% of the cell surface. While the cells double their surface area, the number of nexuses per unit surface remains unchanged (47--48 per 1000 microns2). The packing density of intramembrane particles (or pits) in the nexuses of hypertrophic muscle cells is 6700 . microns-2, which is slightly less than in control muscle cells (7200 . microns-2). A characteristic grouping of the particles (or the pits) within the nexus is often observed. Nexuses between two processes originating from the same cell are common. Nexuses do not occur in the longitudinal muscle.     

Electrical Characteristics of Tunicate Heart Cell Membranes and Nexuses

The tubular ascidian heart is composed of a single layer of cells joined together by apical (zonulae occludentes) and spot (maculae occludentes) nexuses. Intercalated discs or desmosomes were not observed in this tissue. Rectangular pulses of current were applied across the opened and flattened myocardium. Assuming that all the transepithelial current flowed through a uniform gap between cells, the resistivity in the gap must be very high compared to that in bulk solution. It is likely, therefore, that the gap width is of the order of an ionic radius or smaller. Assuming that all the transepithelial current flowed through the cells and that the inner and outer membranes had the same resistivity, the membrane resistivity was about 210 ohms cm² and the membrane capacitance was about 1.6 µF per cm². The myocardial cells were found to be in electrical continuity with each other through the nexuses since current could be passed through a strip of myocardium in a sucrose gap. Assuming that the longitudinal resistance of the cytoplasm was negligible, the cell-to-cell resistivity of the nexuses was 0.2 ohm cm². It is concluded that the nexuses provide a low resistance pathway between cells and a transepithelial barrier.     

Activation of the fast calcium input in the denervated smooth muscle of the cat nictitating membrane

The excitation and contraction features of innervated and sympathetically denervated smooth muscle strips from cat's nictitating membrane have been studied by single sucrose gap arrangement. Increasing of smooth muscle cells sensitivity to drugs were accompanied by elevation of membrane response and the ability to generation of action potentials. Action potentials have been induced by agonists or high potassium concentration in external solution and spontaneously. In innervated muscle action potentials have been evoked as a result of depolarization by high potassium concentration of TEA blockade of potassium conductance. Induced and spontaneously generated action potentials were blocked by organic and inorganic antagonists of potential dependent Ca++ channels. In Ca-free solution action potentials were absent but might be supported by Ba++. Decrease of Na+ had no effect on smooth muscle excitability. It is supposed that activation of potential depended Ca++ channels in smooth muscle cells with pharmaco-mechanical coupling are under influence of sympathetic nerves.     

A STUDY OF NEXUSES IN VISCERAL SMOOTH MUSCLE

Nexuses are described between the smooth muscle cells of the gizzard of the chick and the pigeon, the vas deferens of the mouse and the guinea pig, and the taenia coli of the guinea pig. The nexuses in the gizzard were examined after osmium tetroxide and potassium permanganate had been used as fixatives. Although differences in the dimensions of the unit membranes and the nexuses were noted, the results with the two fixation techniques were complementary. The distribution of nexuses within the smooth muscle tissues examined was uneven. Nexuses were still present in both small and large pieces of tissue incubated in hypertonic solutions at varying temperatures. Other experiments showed that the degree of contraction at the time of fixation did not affect the presence of nexuses in the tissue. These results indicate that nexuses between smooth muscle cells are stable under a variety of conditions.     

Intercellular junctions between circular and longitudinal intestinal muscle layers

Summary Different intercellular relationships between smooth muscle cells are observed in the tunica muscularis externa of the ileum of guinea-pig. a) simple adjoining of the two muscle cells; b) nexus; c) desmosome-like attachment with dense aggregations on both membranes; d) projection from one muscle cell protruding into another muscle cell. All four types are found in the circular muscle layer. On the contrary, in the longitudinal layer clear-cut nexuses are not observed. Simple adjoining of muscle cells, nexuses, and desmosome-like attachments are observed between muscle cells of the circular layer and the longitudinallayer.Dedicated to Professor Angelo Bairati, on the occasion of his sixtieth birthday.I thank Professor E. G. Gray for discussion, Professor J. Z. Young for use of facilities, and the Wellcome Trust for a fellowship.     

Gap junctions of the muscles of the small and large intestine

Summary The distribution of gap junctions (nexuses) in various parts of the small and large intestines of the guinea-pig was studied using the freeze-fracture technique and in thin sections. The percentage area of smooth muscle cell surface occupied by gap junctions varies from 0.50% in the circular muscle of the duodenum to zero in the longitudinal muscle of the ileum. In the circular muscle of the jejunum and ileum the area occupied by nexuses is 0.22% (or about 11 m² per cell). The sizes of junctions range from less than 0.01 m² to 0.20 m², with two-thirds of them being smaller than 0.05 m². In the colon, gap junctions are rare, very small and confined to the circular muscle layer. Even the smallest aggregates of intramembrane particles correspond to areas of close apposition between the membranes of adjacent cells; it is therefore justified to interpret them as being gap junctions. Some gap junctions are formed between a smooth muscle cell and an interstitial cell. Gap junctions are not found in the longitudinal muscle of the small intestine; this is in sharp contrast to the abundance of gap junctions in the adjacent circular layer.In the small intestine of cats and rabbits, gap junctions are abundant in the circular muscle layer, whereas they are very small in size and very few in number in the longitudinal muscle layer.The authors wish to thank Mr Peter Trigg and Miss Eva Franke for help and support. This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

Physiological properties of the penis retractor muscle of Aplysia

The properties of the penis retractor muscle of Aplysia have been studied using intracellular, sucrose gap and tension recording. The fibers are of the invertebrate smooth muscle type and exhibit slow contractions which occur spontaneously or in response to stretch in isolated preparations. Individual muscle fibers are innervated by excitatory and inhibitory axons. A variety of sizes of excitatory and inhibitory junctional potentials can be recorded from them. The innervation is probably diffuse and functionally polyneuronal. The fibers are electrically coupled, permeable to potassium and chloride at rest, and exhibit no overshooting active responses. The muscle shows graded responses of depolarization and contraction proportional to strength of nerve stimulation. Facilitation and depression of junctional potentials are seen with various frequencies of nerve stimulation. Post-tetanic potentiation occurs with nerve stimulation at frequencies from 2 to 50 Hz and is suppressed in the presence of increased extracellular calcium concentrations.     

FURTHER OBSERVATIONS ON THE OCCURRENCE OF NEXUSES IN BENIGN AND MALIGNANT HUMAN CERVICAL EPITHELIUM

An estimate is made of the frequency of occurrence of nexuses ("gap junctions") in a spectrum of human cervical epithelia, ranging from normal to malignant, since a deficiency of nexuses may be important in abnormal cell-to-cell communication in malignant tissues. The normal cervical epithelium has approximately ten nexuses per cell in the basal layer of proliferating cells and 200 nexuses per cell in the more differentiated intermediate zone. Nexuses are rare between invasive malignant epithelial cells (carcinoma cells). In many areas of cell proliferation near the edge of the tumor mass, fewer than one nexus per cell is present. However, up to four nexuses per cell can be found in some well differentiated regions of invasive carcinoma. Preinvasive malignant epithelia (severe dysplasia and carcinoma-in situ) have as few nexuses as invasive carcinoma. In abnormal but benign epithelia (squamous metaplasia and mild dysplasia), nexuses are abundant. The data indicate that a decrease in number of nexuses correlates with the severity of the morphological alteration in the dysplastic epithelium. Also the deficiency of nexuses in groups of carcinoma cells can occur many cell generations before the development of invasion of the malignant epithelium into the connective tissue. The diminution of nexuses before invasion suggests that a deficiency of nexuses may be one of the important factors in eventually permitting the development of the diffusely infiltrating type of invasion which is characteristic of highly malignant tumors such as squamous carcinomas.     

The correction factors for sucrose gap measurements and their practical applications.

The distribution of extracellular and intracellular potential in the sucrose gap apparatus, previously established for a single fiber using the cable equations for a core conductor model (Jirounek and Straub, Biophys. J., 11:1, 1971), is obtained for a multifiber preparation. The exact equation is derived relating the true membrane potential change to the measured potential differences across the sucrose gap, the junction potentials between sucrose and physiological solution, the membrane potential in the sucrose region, and the electrical parameters of the preparation in each region of the sucrose gap. The extracellular potential distribution has been measured using a modified sucrose gap apparatus for the frog sciatic nerve and the rabbit vagus nerve. The results indicate a hyperpolarization of the preparations in the sucrose region, of 60--75 mV. The hyperpolarization is independent of the presence of junction potentials. The calculation of the correction terms in the equation relating the actual to the measured potential change is illustrated for the case of complete depolarization by KC1 on one side of the sucrose gap. The correction terms in the equation are given for various experimental conditions, and a number of nomographic charts are presented, by means of which the correction factors can be rapidly evaluated.     

Airway smooth muscle responsiveness from dogs with airway hyperresponsiveness after O3 inhalation

Airway hyperresponsiveness occurs after inhalation of O3 in dogs. The purpose of this study was to examine the responsiveness of trachealis smooth muscle in vitro to electrical field stimulation, exogenous acetylcholine, and potassium chloride from dogs with airway hyperresponsiveness after inhaled O3 in vivo and to compare this with the responsiveness of trachealis muscle from control dogs. In addition, excitatory junction potentials were measured with the use of single and double sucrose gap techniques in both groups of dogs to determine whether inhaled O3 affects the release of acetylcholine from parasympathetic nerves in trachealis muscle. Airway hyperresponsiveness developed in all dogs after inhaled O3 (3 ppm for 30 min). The acetylcholine provocative concentration decreased from 4.11 mg/ml before O3 inhalation to 0.66 mg/ml after O3 (P less than 0.0001). The acetylcholine provocative concentration increased slightly after control inhalation of dry room air. Airway smooth muscle showed increased responses to both electrical field stimulation and exogenous acetylcholine but not to potassium chloride in preparations from dogs with airway hyperresponsiveness in vivo. The increased response to electrical field stimulation was not associated with a change in excitatory junctional potentials. These results suggest that a postjunctional alteration in trachealis muscle function occurs after inhaled O3 in dogs, which may account for airway hyperresponsiveness after O3 in vivo.     

Freeze-fracture studies of nexuses between smooth muscle cells. Close relationship to sarcoplasmic reticulum

The freeze-fracture appearance of the nexus was compared in the smooth muscle of guinea pig sphincter pupillac, portal vein, pulmonary artery, taenia coli, uretzr, and vas diferens, mouse vas deferens, chicken gizzard and anterior mesenteric artery, and toad stomach. Nexuses are particularly numerous in the guinea pig sphincter pupillae; they are usually oval and their average area is 0.15 mum2, although some as large as 0.6 mum2 were seen. Small aggregations of particles were observed which would not be recognizable as nexuses in thin section. What constitutes the minimum size of a nexus is discussed. It is estimated that the number of nexuses per cell in this preparation is of the order of tens rather than hundreds. All nexuses examined had 6-9-nm particles in the PF face, with corresponding 3-4-nm pits on the EF face forming a polygonal tending towards a hexagonal lattice. The nexuses are arranged in rows parallel to the main axis of the cell, usually alternating with longitudinal rows of plasmalemmal vesicles. Many nexuses in the guinea pig sphincter pupillae, chicken gizzard, and toad stomach show a close relationship with sarcoplasmic reticulum. The possibility that this may have some role in current flow across this specialized junction is discussed.     

Action potentials in gastrointestinal smooth muscle.

Recent investigation of the ultrastructure and electrophysiology of gastrointestinal smooth muscle layers has revealed a fascinating heterogeneity in cell type, cell structure, intercellular communication, and generated electrical activities. Networks of interstitial cells of Cajal (ICC) have been identified in many muscle layers and evidence is accumulating for a role of these networks in gut pacemaking activity. Synchronized motility in the organs of the gut result from interaction between ICC, neural-tissue, and smooth muscle cells. Regulation of cell to cell communication between the different cell types will be an important area for further research. Progress has been made in the elucidation of the ionic basis of the slow wave type action potentials and the spike-like action potentials. The mechanism underlying smooth muscle autorhythmicity seems different from that encountered in cardiac tissue, and evidence exists for metabolic regulation of the frequency of slow wave type action potentials.     

Hypertrophy of intestinal smooth muscle

Proximal to an experimental stenosis of the small intestine of rats and guinea-pigs a remarkable hypertrophy of the muscle coat develops 3-5 weeks after the operation. There is no increase in the length of the intestine but an overall increase in volume of the muscularis externa up to 10 times. This increase is accounted for by an increase in size and in number (by mitosis) of smooth muscle cells of both the longitudinal and circular layers. Bundles of newly-formed smooth cells appear in the serosa and are circularly arranged. In the hypertrophic smooth muscle cells of the circular layer the ratio of surface to volume is 0.80 (0.80 mum2 of cell surface for every mum3 of cell volume) as against 1.4 in the control muscle. The hypertrophic muscle cells have a highly developed sarcoplasmic reticulum and show a large number of nexuses. The density of innervation (number of axons per given number of smooth muscle cells) is smaller than in controls. Few collagen fibrils are visible in the extracellular space.     

Expression of connexin43 gap functions between cultured vascular smooth muscle cells is dependent upon phenotype

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.     

Deficiency of intramuscular ICC increases fundic muscle excitability but does not impede nitrergic innervation

The motility of the gastrointestinal tract is generated by smooth muscle cells and is controlled to a large extent by an intrinsic neural network. A gap of approximately 200 nm usually separates nerve varicosities from smooth muscle cells, which suggests that direct innervation of the smooth muscle by synapses does not occur. Enteric nerves do make synapse-like contact with proposed regulatory cells, the interstitial cells of Cajal (ICC), which in turn may be in gap junction contact with smooth muscle cells. The role played by ICC in enteric innervation is controversial. Experimental evidence has been presented in vitro for the hypothesis that nitrergic inhibitory innervation is strongly reduced in the absence of ICC. However, in vivo data appear to dispute that. The present report provides evidence that explains the discrepancy between in vivo and in vitro data and provides evidence that inhibitory neurotransmitters can reach smooth muscle cells without hindrance when ICC are absent. The fundic musculature shows increased responses to substance P-mediated innervation and shows marked spontaneous activity, which is consistent with increased muscle excitability.     

Detrusor smooth muscle cells of the guinea-pig are functionally coupled via gap junctions in situ and in cell culture

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.     

Is the nexus necessary for cell-to-cell coupling of smooth muscle?

Summary Electronmicroscopic study of electrically coupled smooth muscles was undertaken to determine the distribution of nexuses in various types of smooth muscle. The study revealed that while nexal structures were commonplace in some types of smooth muscle, they were very rare or absent in others, even though in some cases these cells were only a few nanometers distant from one another. The persistence in thin section of these structures in the main circular muscle of dog intestine after poor fixation, fixation under strain, cell shrinkage, and metabolic damage of various sorts seems to rule out the thesis that they are labile. The absence of nexuses in longitudinal muscle of dog intestine examined both by thin section and by freeze fracture suggests that in this tissue they are absent or very rarein vivo and cannot account for electrical coupling.Nexuses were discernible in thin sections of main circular muscle after a variety of experimental conditions of fixation. Metabolic inhibition orin vitro permanganate fixation partially destroyed nexal contacts. These procedures induced tissue, membrane apposition and an accompanying increase in the number of structures which resemble nexuses at low magnification (nexus-like structures). Nexus-like structures occurred in all smooth muscle fixed byin vitro permanganate associated with apposition of membranes and poor preservation of basement membrane. A technique ofin vitro permanganate fixation was developed which prevented tissue swelling; consequently nexus-like structures were absent in tissues so treated. The suggestion is made that some structures described in the literature as nexuses, following permanganate fixation, may represent nexus-like structures.The balance of evidence suggests that nexuses need not be present for electrical coupling of some smooth muscle cells, in which other types of cell-to-cell contacts must be invoked.