Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

1992 ◽  
Vol 50 (1) ◽  
pp. 706-707
Author(s):  
Ji-da Dai ◽  
M. Joseph Costello ◽  
Lawrence I. Gilbert
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Manduca Sexta ◽  
Freeze Fracture ◽  
Cell Communication ◽  
Cellular Level ◽  
Thin Sections ◽  
Prothoracic Glands ◽  
Polyhydroxylated Steroids ◽  
Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

scholarly journals Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures.

1982 ◽  
Vol 92 (1) ◽  
pp. 183-191 ◽  
Author(s):  
D M Larson ◽  
J D Sheridan
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Tight Junctions ◽  
Lucifer Yellow ◽  
Primary Cultures ◽  
Freeze Fracture ◽  
Cell Transfer ◽  
Isolated Cells ◽  
Thin Sections ◽  
Cell To Cell Transfer

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.

scholarly journals A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators

2021 ◽  
Vol 22 (3) ◽  
pp. 1417
Author(s):  
Azeem Danish ◽  
Robin Gedschold ◽  
Sonja Hinz ◽  
Anke C. Schiedel ◽  
Dominik Thimm ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
High Throughput Screening ◽  
Cell Communication ◽  
Receptor Activation ◽  
Adenosine A2a Receptor ◽  
Intracellular Camp ◽  
Donor Cells ◽  
A2a Receptor ◽  
Adenosine A2a

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Organization and synaptic ultrastructure of glomeruli in the antennal lobes of the moth Manduca sexta : a study using thin sections and freeze-fracture

1981 ◽  
Vol 213 (1192) ◽  
pp. 279-301 ◽  
Keyword(s):  
Manduca Sexta ◽  
Antennal Lobe ◽  
Freeze Fracture ◽  
Central Area ◽  
Presynaptic Membrane ◽  
Thin Sections ◽  
Large Numbers ◽  
Synaptic Ultrastructure ◽  
Large Particles ◽  
Antennal Nerve

The antennal lobe of the brain of Manduca sexta comprises a central area of coarse neuropil surrounded by dense, spheroidal glomeruli, where all synaptic interactions between antennal-nerve axons and the second-order neurons of the lobe occur. Neuronal interactions in the glomeruli are complex, involving several types of neuritic profiles and mediated by synapses with a one-to-many ratio of pre- to postsynaptic elements. Presynaptic profiles in the glomeruli have been categorized into three types, containing round clear vesicles, large numbers of large dense-cored vesicles, and pleiomorphic clear vesicles, respectively. Preliminary studies of horseradish peroxidase-filled axons and neurons indicate that antennal-nerve axons form synapses without large numbers of dense-cored vesicles and that antennal-lobe neurons not only receive synapses but also may synapse onto other elements in the antennal lobe. A typical synaptic contact involves multiple postsynaptic elements apposed in pairs to an individual presynaptic element. The presynaptic element contains a bar-shaped membrane-associated density, which follows a shallow groove in the membrane and is flanked by synaptic vesicles. Postsynaptic elements are lined by membrane-associated densities in the region opposite to the synaptic bar, and may be observed to participate in serial synapses. Freeze-fracture replicas of the glomerular neuropil contain many membrane specializations that are thought to be presynaptic, some of which resemble those of vertebrate excitatory synapses. At these apparently presynaptic regions, large particles cluster in the P face of the membrane and are often surrounded by plasmalemmal deformations presumably representing sites of exo- or endocytosis. The shape of the predominant type of presynaptic membrane specialization (a plaque) does not match the shape of the presynaptic membrane-associated density (a bar); this raises the possibility that vesicle release occurs at isolated ‘active zones’ along the presynaptic bar. Postsynaptic sites are represented by clusters of large particles in the E face of the postsynaptic membrane.

The syneretic lens junction

1984 ◽  
Vol 42 ◽  
pp. 16-19
Author(s):  
J. David Robertson ◽  
M.J. Costello ◽  
T.J. McIntosh
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Membranes ◽  
Freeze Fracture ◽  
Enzymatic Digestion ◽  
Fiber Cell ◽  
Intermembrane Space ◽  
Minor Components ◽  
Thin Sections ◽  
Fiber Cells

The lens of the eye consists of closely adherent greatly elongated flattened narrow fiber cells that are electrically coupled by gap junctions. In thin sections the 100-150 Å intermembrane space usually seen in tissues between adjacent cells is greatly reduced between adjacent fiber cells. Freeze-fracture-etch (FFE) studies have demonstrated gap junctions between fiber cells. Several workers have observed expanses of square crystallinity in fiber cell membranes with a lattice constant of 6-7 nm. This has usually been attributed variously to artifact induced by calcium, pH or proteolytic enzymatic digestion. Square arrays have been seen in isolated fractions of fiber cell membranes prepared with detergents as minor components and dismissed as relatively insignificant and either related or unrelated to gap junctions. Some have regarded them as a form of gap junction.

Physiology of Insect Rhythms

1974 ◽  
Vol 60 (2) ◽  
pp. 371-382 ◽  
Author(s):  
JAMES W. TRUMAN ◽  
LYNN M. RIDDIFORD
Keyword(s):  
Developmental Stage ◽  
Manduca Sexta ◽  
Prothoracic Gland ◽  
Dorsal Midline ◽  
Prothoracic Glands ◽  
The Times ◽  
Manduca Sexta Larvae ◽  
The Brain ◽  
Pink Pigment ◽  
Stimulation Of

1. Late in the fifth instar, Manduca sexta larvae cease feeding and become ‘wandering larvae’ which are morphologically characterized by an ‘exposed heart’ and the appearance of a pink pigment along the dorsal midline. Two days later ocellar retraction signals the beginning of the prepupal period and 3 days thereafter the pupal ecdysis occurs. 2. The timing of the endocrine events which are responsible for these changes was determined by ligaturing animals of the appropriate developmental stage at various times of day. The times of prothoracicotropic hormone (PTTH) release by the brain were determined by neck ligations. Estimates of the times of prothoracic gland activity were obtained through the isolation of abdomens. 3. It was found that the fifth stage larva releases PTTH on two occasions. The first release lasts approximately 3.5 h and triggers the transformation to the wandering stage. The second release occurs two days later, lasts at least 7 h, and provokes the onset of the pupal moult. 4. The prothoracic glands are involved in triggering the same two changes. In the first instance the glands apparently require the continuing influence of the brain and consequently secrete for about 3.5 h. During the stimulation of the pupal moult the prothoracic glands become ‘turned-on’ and continue to secrete for at least 10 h after the time when the brain is no longer required. In this latter instance the total time of prothoracic gland activity may be as long as 17 h.

Plasma membranes, cell junctions and cuticle of the rectal chloride epithelia of the larval dragonfly Aeshna cyanea

1983 ◽  
Vol 59 (1) ◽  
pp. 159-182
Author(s):  
J. Kukulies ◽  
H. Komnick
Keyword(s):  
Gap Junctions ◽  
Structural Control ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Septate Junction ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Apical Region ◽  
Thin Sections ◽  
Aeshna Cyanea

The cell membranes and cell junctions of the rectal chloride epithelia of the larval dragonfly Aeshna cyanea were examined in thin sections and by freeze-fracture. These epithelia function in active ion absorption and maintain a high concentration gradient between the haemolymph and the fresh-water environment. Freeze-fracturing reveals fine-structural differences in the intramembraneous particles of the luminal and contraluminal plasma membranes of these epithelia, reflecting the functional diversity of the two membranes, which are separated by the junctional complex. The particle frequency of the basolateral plasma membranes is reduced after transfer of the larvae into high concentrations of environmental salinity. The junctional complex is located in the apical region and composed of three types of cell junctions: the zonula adhaerens, seen in freeze-fracture as a nearly particle-free zone; the extended and highly convoluted pleated septate junction and randomly interspersed gap junctions of the inverted type. Gap junctions also occur between the basolateral plasma membranes. They provide short-cuts in the diffusion pathway for direct and rapid co-ordination of the interconnected cell processes. Colloidal and ionic lanthanum tracer solutions applied in vivo from the luminal side penetrate through the cuticle via epicuticular depressions, but invade only the apical portion of the junctional complex. This indicates that the pleated septate junction constitutes a structural control of the paracellular pathway across the chloride epithelia, which are devoid of tight junctions. The structure of the pleated septate junctions is interpreted as a device for the extension of the diffusion distance, which is inversely related to the net diffusion. A conservative estimate of the total length of the junction, and the number and extension of septa reveals that the paracellular route exceeds the transcellular route by a factor of 50.

scholarly journals Low resistance junctions in crayfish. Structural changes with functional uncoupling.

1976 ◽  
Vol 70 (2) ◽  
pp. 419-439 ◽  
Author(s):  
C Peracchia ◽  
A F Dulhunty
Keyword(s):  
Gap Junctions ◽  
Conformational Changes ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Particle Diameter ◽  
Electrical Measurements ◽  
Thin Sections ◽  
Giant Axons ◽  
Intramembrane Particles ◽  
Center Distance

Electrical uncoupling of crayfish septate lateral giant axons is paralleled by structural changes in the gap junctions. The changes are characterized by a tighter aggregation of the intramembrane particles and a decrease in the overall width of the junction and the thickness of the gap. Preliminary measurements indicate also a decrease in particle diameter. The uncoupling is produced by in vitro treatment of crayfish abdominal cords either with a Ca++, Mg++-free solution containing EDTA, followed by return to normal saline (Van Harreveld's solution), or with VAn Harreveld's solution containing dinitrophenol (DNP). The uncoupling is monitored by the intracellular recording of the electrical resistance at a septum between lateral giant axons. The junctions of the same septum are examined in thin sections; those of other ganglia of the same chain used for the electrical measurements are studied by freeze-fracture. In controls, most junctions contain a more or less regular array of particles repeating at a center to center distance of approximately 200 A. The overall width of the junctions is approximately 200 A and the gap thickness is 40-50 A. Vesicles (400-700 A in diameter) are closely apposed to the junctional membranes. In uncoupled axons, most junctions contain a hexagonal array of particles repeating at a center to center distance of 150-155 A. The overall width of the junctions is approximately 180 A and the gap thickness is 20-30 A. These junctions are usually curved and are rarely associated with vesicles. Isolated, PTA-stained junctions, also believed to be uncoupled, display similar structural features. There are reasons to believe that the changes in structure and permeability are triggered by an increase in the intracellular free Ca++ concentration. Most likely, the changes in permeability are caused by conformational changes in some components of the intramembrane particles at the gap junctions.

scholarly journals Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion.

1979 ◽  
Vol 82 (2) ◽  
pp. 441-448 ◽  
Author(s):  
P Meda ◽  
A Perrelet ◽  
L Orci
Keyword(s):  
Insulin Secretion ◽  
B Cells ◽  
Gap Junctions ◽  
Freeze Fracture ◽  
Secretory Activity ◽  
Isolated Rat Islets ◽  
Pancreatic B Cells ◽  
Stimulation Of

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.

Nonrandom distribution of gap junctions between pancreatic beta-cells

1980 ◽  
Vol 238 (3) ◽  
pp. C114-C119 ◽  
Author(s):  
P. Meda ◽  
J. F. Denef ◽  
A. Perrelet ◽  
L. Orci
Keyword(s):  
Insulin Secretion ◽  
Gap Junctions ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Freeze Fracture ◽  
Membrane Area ◽  
Nonrandom Distribution ◽  
Isolated Rat Islets ◽  
Rat Islets Of Langerhans ◽  
Stimulation Of

The numerical and spatial distribution of gap junctions between insulin-containing cells (beta-cells) under resting and stimulated conditions of insulin secretion were quantitatively analyzed in freeze-fracture replicas of isolated rat islets of Langerhans. The results show that the beta-cells located at the periphery of the islet have twice as many gap junctions per unit membrane area as the beta-cells situated in the islet center. In both locations, gap junctions assumed a nonrandom clustering on the beta-cell membranes. During stimulation of insulin secretion, the gap junctions were found increased between the central and between the peripheral beta-cells. The degree of their clustering was also modified. The latter change depended both on the location of the gap junctions in the islet and on the type of stimulation used (high glucose or glibenclamide).

Light and Electron Microscopic Studies Regarding Cell Contractility and Cell Coupling in Light Sensitive Smooth Muscle Cells from the Isolated Frog Iris Sphincter

1987 ◽  
Vol 42 (7-8) ◽  
pp. 977-985 ◽  
Author(s):  
Klaus V. Wolf
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Sphincter Muscle ◽  
Cell Coupling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Cell Contractility ◽  
Microscopic Studies ◽  
Iris Sphincter

(1) In light microscopical studies of living isolated frog irises, it was found that the maximal areas of experimentally light induced contractions in the m. sphincter pupillae were located beneath small illuminated regions. There were no visible contractions of muscle cells outside the illuminated areas. It was shown that exposure to light could directly cause contractions of isolated single sphincter muscle cells. (2) Junctional structures of the iris sphincter cells were studied by means of thin sections and freeze fracture electron microscopy. Intermediate junctions, a few focal tight junctions and occasional small gap junctions were identified. Pit containing intramembranous particles which resemble gap junction connexons were found in large numbers, dispersed over the plasmalemmas of sphincter muscle cells. From these physiological and morphological observations, it is concluded that sphincter muscle cells of the frog iris may be coupled via gap junctions, but that the cell coupling is not sufficiently extensive to form the basis for a functional syncytium.

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