Gap junctional communication in the vibration-sensitive response of sea anemones

Although gap junctions occur in auditory and vestibular systems, their function is unclear. Here we present evidence for gap junctional communication in transmitting mechanosensory signals in a sea anemone model system. Hair bundles on anemone tentacles are vibration-sensitive mechanoreceptors that regulate discharge of nematocyst from effector cells. We find that vibration-dependent nematocyst discharge is selectively and reversibly blocked by the gap junction uncouplers, heptanol and arachidonic acid. Epidermal cells within excised tentacles exhibit a low level of dye coupling which is significantly enhanced upon deflection of overlying hair bundles. Dye coupling is inhibited both by gap junction uncouplers and by agents that interfere with mechanotransduction, including streptomycin and elastase. Electrophysiological data suggest gap junctional communication between cells giving rise to different hair bundles. When hair bundles are stimulated with a sweep of vibrations, individual cells show responses to five to eight frequencies. The number of responsive frequencies is reduced to one or two by heptanol and essentially abolished with streptomycin treatment. Immunoreactivity to the gap junction protein, connexin 43, is abundant in the tentacle epidermis and localized to membranes at junctions between several cell types. Small areas of close membrane apposition are observed between these cell types with intermembrane clefts of 4-7 nm. Of the several membrane proteins isolated from tentacles, immunoreactivity to connexin 43 is observed in a single band with an apparent molecular weight of approximately 46 kDa.     

Connexins and gap junctions in the inner ear

Mutations in the genes for three different isotypes of the gap junction channel protein connexin are associated with deafness. This indicates an important role for gap junctions in auditory function and provides an opportunity to explore structure-function relationships in the connexin molecule. We have been examining the distribution of gap junctions and the pattern of connexin expression in the mature inner ear and during development, and the effect of specific mutations on the processing and functionality of the expressed connexin proteins in an in vitro system.     

内耳间隙连接和连接蛋白基因家族与遗传性聋

耳蜗有非感觉上皮细胞和结缔组织细胞2套间隙连接系统,内耳前庭的感觉上皮也有间隙连接。已发现在内耳表达的连接蛋白有Cx26、Cx30、Cx31、Cx32、Cx43和Cx45。连接蛋白是同一基因家族编码,其中GJB2(Cx26)、GJB3(Cx31)、GJB6(Cx30)、GJA1(Cx43)、GJB1(Cx32)都与遗传性聋有关。GJB2基因敲除小鼠在胚胎期致死。GJB3敲除小鼠60%在胚胎死亡,成活小鼠无耳聋和皮肤疾病等表型。GJA1裸鼠表现心脏发育缺陷与膜内和软骨内成骨延迟。GJB1敲除小鼠表型无明显异常,但外周神经表现脱髓鞘改变。不同间隙连接的缺陷导致听力下降的机制可能不同。间隙连接与耳聋关系的深入研究,可能有助于阐明听觉和耳聋的部分机制。     

Effects of gap junction uncoupling in the gerbil cochlea

OBJECTIVE: To gain insight into molecular and cellular mechanisms regulating cochlear potassium (K+) recycling, including the possible effects of mutations in the gene, which encodes the gap junction protein connexin 26. Intercellular K+ flux was manipulated in vivo by infusion of the gap junction uncoupler proadifen (SKF-525A) into perilymph. Functional and structural alterations induced by gap junction blockade were assessed by electrophysiological and morphologic analysis. STUDY DESIGN: Laboratory study using an animal model. METHODS: Physiological effects of acute and chronic uncoupling of gap junctions in the Mongolian gerbil inner ear were evaluated by measurement of compound action potential (CAP) thresholds and input-output (I/O) functions, distortion product otoacoustic emissions (DPOAE), and the endocochlear potential (EP). Morphologic changes were assessed by electron microscopy. RESULTS: Acute exposures to proadifen resulted in large decreases in EP values, DPOAE magnitudes, and CAP I/O maximum amplitudes and an increase in high-frequency CAP thresholds. These physiological changes were accompanied by vacuolization of type II and type V fibrocytes in the lateral wall of the cochlea. Chronic treatments revealed some recovery in EP values and CAP thresholds, which showed a relatively flat 15- to 20-dB elevation across frequencies. CONCLUSIONS: Gap junctions play a significant role in normal cochlear function. In particular they appear to be essential for maintaining the EP, an activity that could be related to their participation in K+ recycling. Thus, hearing losses associated with mutations in the gene that alter the expression or function of connexin 26 may result from a diminished capacity to recycle K+ from perilymph back to the stria vascularis and a consequent decline in the EP.     

The effect of gentamicin-induced hair cell loss on the tight junctions of the reticular lamina

Freeze fracture has been used to examine junctional complexes within the gentamicin damaged guinea pig organ of Corti during the period of structural reorganisation associated with hak cell loss. When examined up to 28 days after a regime of chronic gentamicin administration, no significant alterations to the pattern or nature of the complex tight junctions associated with the hair cell apex could be detected. In contrast, marked abnormalities of the junctions between adjacent supporting cells, and evidence of formation of new gap junctions on the lateral membranes of Deiters' cell was seen. The use of lanthanum as an electron dense tracer in thin sections also showed that no breakdown of permeability barriers occurred in the reticular lamina. These results support the concept of a controlled response by the organ of Corti, with conservation of the tight junction of the hak cell and maintenance of permeability barriers. Early changes to the membranes of the stereocilia and lateral cistemae of the outer hair cells were also identified. These may indicate direct effects of gentamicin upon membrane structure.     

Freeze fracturing of the human stria vascularis

The stria vascularis is an important functional element in the mammalian cochlea. This special tissue is considered to be the source of the endocochlear potential and thus the driving force for the production of a receptor response to the auditory stimulus. In order to maintain its function, the stria vascularis needs to be separated from the endolymphatic space by a tight seal. This seal is comprised of tight junctions in the marginal cell layer. The junctional arrangement in the stria vascularis is described, utilizing the freeze-fracturing technique which allows the visualization of large expansions of plasma membrane. The marginal cells are generally separated by tight junctions of the moderately tight to tight type. In places, however, even so-called leaky junctions with only a few sealing strands are present. Whereas the intermediate cell layer seems to lack tight junctions, the basal cells are connected by extensive tight junctions more or less covering the entire cell. These junctions seem to form an extremely tight barrier against the spiral ligament. Gap junctions are also present in the tissue. Intermediate cells as well as the basal cells are coupled by gap junctions. In the basal cell layer, gap junctional elements may also be found inside the large tight junctions comprising so-called mixed junctions.     

Scanning electron microscopic study of the neuromuscular junctions of the cricothyroid and thyroarytenoid muscles in rats

Neuromuscular junctions were observed in the cricothyroid (CT) and thyroarytenoid (TA) muscles of adult rats by scanning electron microscopy after removing the intramuscular connective tissue components using the HCI hydrolysis method. Morphologically, the junctions were classified into three types in the CT muscle and two types in the TA muscle, based on the structural characteristics of the subneural apparatuses, including junctional folds. In the CT muscle, type 1 junctions (32%) consisted of more than 15 cup-like depressions with slit-like junctional folds. Type 2 junctions (20%) were characterized by approximately 10 cup-like depressions with a small number of pit- or slit-like junctional folds. Type 3 junctions (48%) had irregular labyrinthine gutters with slit-like junctional folds. In the TA muscle, type 1 (82%) and 2 (18%) junctions had similar structures to type 1 and 2 junctions in the CT muscle, respectively. Histochemical studies using myosin adenosine triphosphatase staining showed that both CT and TA muscles predominantly consisted of type II muscle fibers (78% and 82%, respectively), and that the diameter of type II fibers was larger than that of type I fibers. These findings suggest that the type 2 junction belongs to type I muscle fibers, while both type 1 and type 3 junctions belong to type II fibers, and that the type 3 junction is a structural variation of the type 1 junction. The significance of the structural differences of the subneural apparatuses in the intrinsic laryngeal muscles is discussed briefly.     

Scanning Electron Microscopic Study of the Neuromuscular Junctions of the Cricothyroid and Thyroarytenoid Muscles in Rats

Neuromuscular junctions were observed in the cricothyroid (CT) and thyroarytenoid (TA) muscles of adult rats by scanning electron microscopy after removing the intramuscular connective tissue components using the HCl hydrolysis method. Morphologically, the junctions were classified into three types in the CT muscle and two types in the TA muscle, based on the structural characteristics of the subneural apparatuses, including junctional folds. In the CT muscle, type 1 junctions (32%) consisted of more than 15 cup-like depressions with slit-like junctional folds. Type 2 junctions (20%) were characterized by approximately 10 cup-like depressions with a small number of pit- or slit-like junctional folds. Type 3 junctions (48%) had irregular labyrinthine gutters with slit-like junctional folds. In the TA muscle, type 1 (82%) and 2 (18%) junctions had similar structures to type 1 and 2 junctions in the CT muscle, respectively. Histochemical studies using myosin adenosine triphosphatase staining showed that both CT and TA muscles predominantly consisted of type II muscle fibers (78% and 82%, respectively), and that the diameter of type II fibers was larger than that of type I fibers. These findings suggest that the type 2 junction belongs to type I muscle fibers, while both type 1 and type 3 junctions belong to type II fibers, and that the type 3 junction is a structural variation of the type 1 junction. The significance of the structural differences of the subneural apparatuses in the intrinsic laryngeal muscles is discussed briefly.     

Epithelial cells of nasal mucosa express functional gap junctions of connexin 43

OBJECTIVE: Recent studies have suggested that inositol triphosphate is transmitted through the gap junction of adjacent epithelial cells and communicates the intracellular calcium wave that controls the beating of cilia. Therefore, gap junction-mediated intercellular communication may modulate the metachronal ciliary beating of respiratory mucosa. MATERIAL AND METHODS: The expression and function of gap junctions were evaluated in nasal mucosa using immunofluorescent staining, Western blotting and Lucifer Yellow dye coupling in both whole nasal mucosa and cultured nasal epithelial cells. RESULTS: Among connexins (Cxs) 26, 32 and 43, only Cx43 was found in the nasal mucosa, in both the epithelial cells and submucosa tissue. Using Western blotting, Cx43 protein was found in protein extracts of both whole mucosa and cultured epithelial cells. The Cx43 protein content increased twofold between the 3rd and 6th days of culture. Lucifer Yellow dye, microinjected into single cultured epithelial cells, had spread to the adjacent cells. CONCLUSION: Cx43 is present in nasal mucosa. The expression of Cx43 on epithelial cells increases after culture. The dye-coupling study in epithelial cells indicated that the signal may be transmitted to neighboring cells via a gap junction composed of Cx43. Thus, Cx43 may be involved in the regulation of metachronal ciliary beating in ciliated epithelial cells and growth or differentiation in nasal tissue.     

Zellverbindungskomplexe im Limbus spirale

Summary The spiral limbus of the chinchilla inner ear was investigated using the freeze-fracture method.In the replica we observed that a) the intercellular space between the epithelial cells of the spiral limbus (interdental cells and inner sulcus cells) is completely sealed from the endolymphatic space by extense tight junctions, b) the epithelial cells are joined to each other by gap junctions, as well as the cells of the perilymphatic space.Tight junctions seal the intercellular space of an epithelium towards an adjoining lumen, gap junctions represent an area of electrotonical and metabolical communication of cells without participation of the intercellular space.From these findings in accordance with previous investigations of the vascular stria, the Reissner-membrane, and the organ of Corti we conclude, that in the inner ear the tight junctions occur independently on location and function of the single epithelium and that they play a role in the maintenance of the endocochlear potential and the high potassium concentration gradient between endolymph and perilymph.The presence of numerous and extense gap junctions between the cells of the perilymphatic space is similar in the vascularised areas of the cochlear duct. A continuous supply of cells of the individual tissue seems to be maintained as well as its participation at the homoeostasis of the surrounding liquid.     

Epithelial Cells of Nasal Mucosa Express Functional Gap Junctions of Connexin 43

Objective—Recent studies have suggested that inositol triphosphate is transmitted through the gap junction of adjacent epithelial cells and communicates the intracellular calcium wave that controls the beating of cilia. Therefore, gap junction-mediated intercellular communication may modulate the metachronal ciliary beating of respiratory mucosa. Material and Methods—The expression and function of gap junctions were evaluated in nasal mucosa using immunofluorescent staining, Western blotting and Lucifer Yellow dye coupling in both whole nasal mucosa and cultured nasal epithelial cells. Results—Among connexins (Cxs) 26, 32 and 43, only Cx43 was found in the nasal mucosa, in both the epithelial cells and submucosa tissue. Using Western blotting, Cx43 protein was found in protein extracts of both whole mucosa and cultured epithelial cells. The Cx43 protein content increased twofold between the 3rd and 6th days of culture. Lucifer Yellow dye, microinjected into single cultured epithelial cells, had spread to the adjacent cells. Conclusions—Cx43 is present in nasal mucosa. The expression of Cx43 on epithelial cells increases after culture. The dye-coupling study in epithelial cells indicated that the signal may be transmitted to neighboring cells via a gap junction composed of Cx43. Thus, Cx43 may be involved in the regulation of metachronal ciliary beating in ciliated epithelial cells and growth or differentiation in nasal tissue.     

Gap junctions in laryngeal carcinoma

Summary The ultrastructural morphology of gap junctions in malignant keratinocytes of invasive squamous cell carcinoma of the human larynx was studied by electron microscopy. The ultrastructure of gap junctions in the malignant keratinocytes was similar to the typical intercellular gap junctions described in normal tissues. In human laryngeal carcinoma, however, the gap junctions were noticeably reduced in number as compared to normal epithelia.Spherical intracytoplasmic structures limited by gap junctional membranes were also found. These annular gap junctions arise by invagination of the cell surface membrane. The loss of gap junctions in laryngeal carcinoma is probably one of the many prerequisites which facilitate tumor invasion.This work was supported by the Fonds Österreichischer Krebsforschungsinstitute and by the Krebsausschuß der Medizinischen Fakultät der Universität Wien     

Formation of junctional complexes in otocysts developed in vitro. A freeze-fracture study

The thirteenth gestational day inner ear anlage (otocyst) was explanted to an in vitro system and cultured for 8 days, i.e. to a time corresponding to birth. The freeze fracturing technique was used to evaluate morphological differentiation, particularly as regards cell membrane specializations. The epithelial cells were found fully differentiated, e.g. there were regularly arranged stereocilia on the hair cells. The development of tight junctions and gap junctions followed the same pattern as in vivo, but tight junctions did not reach the same degree of regularity and maturation as they did in vivo. There were very few gap junctions in our in vitro specimens. Several tight junctions had an odd appearance, with loss of the normally punctate structure of the strands and areas with considerable thickening. It is possible that this specific morphology can be explained by differentiation in the in vitro system.     

Motor end-plate distribution in the human lateral cricoarytenoid muscle

Knowledge of the neuromuscular junction distribution in the intrinsic laryngeal muscles has potential clinical application in directing procedures that affect the function of these muscles through their neuromuscular junctions. We used histochemical techniques and computer graphics to determine the three-dimensional distribution of the motor end-plates in the human lateral cricoarytenoid muscle. In contrast to the results that have been reported for the human thyroarytenoid, cricothyroid, and posterior cricoarytenoid muscles, where end-plates are more diffusely distributed, the results of our study indicate that in the human lateral cricoarytenoid muscle, the neuromuscular junctions are generally found within a broad band at the midlength of the muscle. This more focused distribution should be advantageous clinically in facilitating the manipulation of the motor end-plates in this muscle.     

Freeze-etch visualizations of some elements in the stria vascularis

A quick-freeze, deep-etch study was made to observe the three-dimensional aspect of both membranes and organelles in the stria vascularis of the guinea pig. Our replicas showed various membrane characteristics of the vesicles, e.g., a clathrin basket on the inner surface membrane, aggregation of membrane particles on the protoplasmic fracture face, and regularly arranged surface protuberances on the outer surface membrane. In the cell processes of marginal cells, there were not only bundles of microtubules but also another type of fibril. Many gap junctions were observed between marginal, intermediate, and basal cells. In some replicas, the inner surfaces of the gap junctions were covered with particle protrusions with some ordered arrangements. The functional roles of these elements in the stria vascularis are discussed.     

Three-dimensional distribution of neuromuscular junctions in human cricothyroid

Microinjections of myoneural blocking agents are effective in the treatment of strabismus and have been proposed as a possible technique to treat spastic dysphonia. The success of such a technique would rely on a precise knowledge of the neuromuscular junction distribution in the laryngeal muscle to be injected. In view of the possibility of an involvement of the cricothyroid muscle in spastic dysphonia or other neuromuscular disorders, we determined the three-dimensional distribution of neuromuscular junctions in the human cricothyroid muscle. Cricothyroid muscles obtained from autopsy cases were fixed, sectioned, and processed for the histochemical localization of neuromuscular junction acetylcholinesterase. Using serial sections and a computer interfaced X-Y digitizer, the neuromuscular junctions were referenced to various anatomic landmarks and the neuromuscular junction distribution and reconstructed in three dimension using computer graphics.     

The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency

Ultrastructural analysis of cells in the cochlea's lateral wall was undertaken to investigate morophologic features relevant to the route of K⁺ cycling from organ of Corti (OC) to stria vascularis (StV) and to the question of a transcellular versus an extracellular path. The fine structure of outer sulcus cells (OSCs) evidenced their capacity for uptake of K⁺ from Claudius cells and from perilymph in inferior spiral ligament. Plasmalemmal amplification and mitochondrial density together with known content of Na,K-ATPase testified to activity of type II, IV and V fibrocytes in resorbing K⁺. Location and fine structure afforded a basis for distinguishing subtypes among the type I, II and IV cells. The type 11, IV and V fibrocytes can be viewed as drawing K⁺ from surrounding perilymph and from OSCs and generating an intracellular downhill diffusion gradient for K⁺ flow through gap junctions to subtype Ib and la fibrocytes and strial basal cells. Pumping action enabled by extreme structural specialization of type II fibrocytes is considered to mediate K⁺ translocation across the interruption between the gap junction connected epithelial and gap junction connected fibrocyte systems and to explain ion flow directed toward StV through OSCs and fibrocytes despite their lack of polarity. The OSC bodies shrank, their root bundles expanded and the gap junction contact between OSCs and Claudius cells increased toward the base of the cochlea. Expanding root bundles and type I and IIb fibrocyte populations contrasted with shrinking OHCs and Deiters and tectal cells from the apex to the base of the cochlea. These differences indicated an increased magnitude and alternate route of K+ transport toward the StV in high as compared to low-frequency regions. The augmented K⁺ transport through spiral ligament in basal cochlea correlates with and provides a possible basis for the larger endocochlear potential in the base. The findings appear consistent with current flow extracellularly through scalae tympani and vestibuli and transcellularly through OC, OSCs and class I, II, IV and V fibrocytes.     

The ultrastructure of the supporting system in the guinea pig semicircular canal

Summary The ultrastructure of the perilymphatic space of the guinea pig semicircular canal was examined, using thin sectioning, scanning electron microscopy and freeze-fracture methods. The perilymphatic space was divided into two parts: an area of fibrous matrix and an area of fibroblasts with bundles of fine filaments. In the area of fibroblasts, cell processes and filament bundles formed a meshed pattern. The cell processes were interconnected with each other by both intermediary and gap junctions. The inner surface of the osseous semicircular canal was covered with multilayers of cell processes of both fibroblasts and mesothelial cells. The fact that tight and gap junctions were observed between the cell processes suggests a functional significance in these structures.