Gap junction structure and cell-to-cell coupling regulation: is there a calmodulin involvement?

In most tissues neighboring cells communicate directly with each other by exchanging ions and small metabolites via cell-to-cell channels located at the intermembrane particles of gap junctions. Evidence indicates that the channels close when the [Ca2+]i or [H+]i increases. The channel occlusion (cell-to-cell uncoupling) is mainly a safety device by which cells can isolate themselves from damaged neighboring cells ("healing-over" process). Despite our knowledge of uncoupling agents, the uncoupling mechanism is still poorly understood. Uncoupling treatments have been shown to cause structural changes in gap junctions, characterized by an increase in tightness and regularity (crystallization) of particle packing and a decrease in particle size. Recently these changes have been shown to be induced by Ca2+ or H+ in isolated lens junctions and by Ca2+ in liver junctions, which suggests a close relationship between structural changes and uncoupling, but preliminary studies indicate that the junctional changes may not be synchronous with uncoupling but may lag behind it. However, recent X-ray diffraction data show that the channels of crystalline gap junctions (typical of uncoupled cells) are indeed closed, because they are inaccessible to sucrose (a gap junction permeant). Thus it seems that crystalline junctions are indeed in a non-permeable state, but the occlusion of the channels may precede the crystallization process. In the lens, junction crystallization is inhibited by a calmodulin (CaM) inhibitor, trifluoperazine (TFP). Is CaM involved in the uncoupling mechanism? To test this hypothesis, TFP and calmidazolium (CDZ), the most specific CaM inhibitor, were used on amphibian embryonic cells electrically uncoupled by CO2. Both TFP and CDZ effectively protect the cells from uncoupling, which suggests that CaM participates in the process. As a hypothesis, we propose that channel occlusion follows a CaM-mediated conformational change in the junctional protein. Particle crystallization may follow the conformational changes and result from a modification in electrostatic repulsion among the particles.     

Gap junction dynamics: reversible effects of divalent cations

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode''s solutions containing a Ca++ ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.     

Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.     

Gap junctions. Structural changes after uncoupling procedures

The freeze-fracture appearance of rat stomach and liver gap junctions changes after uncoupling procedures such as inhibition of the metabolism of perfusion with hypertonic sucrose. In control stomach, either fixed immediately or kept for 1 h in a well-oxygenated Tyrode's solution at 37 degrees C, most gap junctions between mucous cells contain particles irregularly packed at an average center-to-center spacing of 10.3-10.5 nm. After 1-h treatment with 2,4-dinitrophenol (DNP), at the same temperature and oxygenation, most particles aggregate hexagonally at an average spacing of approximately 8.5 nm. Similar changes are seen in hypoxic specimens. In control liver, fixed by perfusion, most junctional particles are irregularly packed at an average center-to-center spacing of approximately 10 mm. Small areas of fairly regular hexagonal packing are occasionally seen, where the average particle spacing is 9.2-9.5 nm. In hypoxic liver, the junctional particles form regular hexagonal packings in which the average center-to-center particle spacing is approximately 8.5 nm. In liver perfused with hypertonic sucrose-calcium solutions, following EDTA solutions, most junctions are pulled apart. The separated junctional membranes, expected to be highly impermeable, contain particles regularly and tightly packed as in hypoxic or DNP-treated junctions. Preliminary measurements indicate also a possible change in particle diameter, from approximately 8.6 nm (control) to approximately 7.7 nm (treated). The structural changes are similar to those previously reported in crayfish and may reflect conformational changes in particle subunits resulting in functional uncoupling.     

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.     

Gap junctions and rhombic particle arrays in the freeze-fractured mouse oocyte-follicle cell complex

Intact follicles as well as defolliculated oocytes of the mouse were studied by freeze-fracture electron microscopy. In intact follicles the oocyte plasma membrane shows two prominent types of intra-membrane particle array:gap junctions and yet undescribed rhombic particle arrays. The gap junctions vary in size (from 5 to 500 IMPs) and shape. Occasionally they are organized in so-called formation plaques. The rhombic particle arrays consist of 25 IMPs on an average, the IMP diameter is 10.5 nm, the mean IMP distance is 19.8 nm and the acute angle in the array is 81.3 degrees. After defolliculation the gap junctions disassemble and change transiently into linear IMP arrays. The rhombic particle arrays persist indicating that they are of a non-junctional nature. The possible function of the rhombic particle arrays is discussed in relation to similar membrane specializations in excitable cells.     

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

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.     

Square arrays and their role in ridge formation in human lens fibers

Square arrays in human lens fibers were studied with freeze-fracture and thin-section TEM. In superficial fibers a number of patches of square array particles in the P face and pits in the E face are found in the smooth membrane. In the deeper cortex and the nucleus, fiber cells have undulating membranes and many ridges. Numerous patches of the particles (P face) are distributed in the concave regions, and the pits (E face) in the convex areas of the bumpy membrane. In most ridges, patches of the particles occur at regular intervals in the "valley" portion, while the pits are on the "crest" portion of ridges. Also, continuous square arrays having the same "valley" location as the regularly arranged patches are found in areas with extensive ridge patterns. The overlapping of the outer portions of two adjacent square arrays is found on the sides between the "crest" and the "valley" of the ridges. Structurally, square arrays are located in a nonjunctional part of the membrane; in an orthogonal crystalline arrangement; and with a particle size of about 6 nm and center-center spacing about 6.4 nm. They are structurally different from gap junctions found in the lens fibers. Thin-section studies reveal two types of cellular contacts: thin pentalamellar structures (about 12-13 nm in overall thickness) associated with the ridge patterns are believed to be square arrays; thick heptalamellar structures (about 16-17 nm in overall thickness) with a narrow gap in between the two central laminae are believed to be gap junctions. This study strongly suggests that square arrays are specifically involved in ridge formation in human lens fibers.     

Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pH(i)), did not exhibit the pH(i)-regulatory HCO(3)(-)/Cl(-) and Na(+)/H(+) exchange activities found in fully-grown oocytes, and had low pH(i). However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pH(i) increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pH(i) is instead maintained at approximately 7.2 throughout oocyte development, and the growing oocyte exhibits HCO(3)(-)/Cl(-) exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO(3)(-)/Cl(-) exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pH(i) may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO(3)(-)/Cl(-) exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO(3)(-)/Cl(-) exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pH(i) is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.     

Morphometric analysis of gap junctions in rat myocardium after hyperkalemia

The effect of membrane depolarization was investigated on gap junctions from isolated rat hearts perfused with a modified Krebs-Henseleit solution containing 16 mM K+. After freeze-fracturing, the configuration of the junctional particles in the ventricular myocardium was analysed by measurements of connexon densities and centre-to-centre distances between neighbouring particles. Both in control and hyperkalemic tissue, the gap junctions occur on the intercalated discs as round or oval aggregates of connexons which are closely and regularly packed in small, criss-cross-oriented arrays separated by particle-free aisles. Within the arrays, the mean (+/- SD) centre-to-centre distances between particles from control and hyperkalemic tissue, i.e. 9.17 +/- 1.52 and 9.15 +/- 1.51 mm, respectively, are not significantly different. Similarly, comparison of particle densities after control and high-K+ perfusion, i.e. 8,490 +/- 600 and 8,420 +/- 620 particles/microns 2, respectively, reveals no difference in the proportion of the particle arrays to the empty aisles. The apparently unaltered gap junctional morphology after depolarization of the sarcolemma by high-K+ perfusion provides support for the electrophysiological finding that the conductance of cardiac gap junctions is insensitive to membrane potential.     

Low resistance junctions in crayfish. Structural changes with functional uncoupling

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.     

The effect of K+/H+ antiporter nigericin on gap junction permeability

The K+/H+ antiporter nigericin inhibits the intercellular exchange of the fluorescent dye Lucifer Yellow between DM15-transformed fibroblasts derived from the Djungarian hamster. The efficacy of nigericin action was related to its concentration and time of incubation. The nigericin-induced uncoupling effect on gap junctions was reversible and was shown to be based on its ability to cause cystolic acidification. The effect of nigericin on dye-coupling in intact and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) pretreated cells did not differ, indicating that the uncoupling effect of H⁺ on gap junctions in DM15 cells was not mediated by the TPA-dependent isoform of protein kinase C.Abbreviations: BCECF, 2,7-bis-(2-carboxyethyl)-5(6)carboxyfluoresceine - BS, bovine serum - LY, Lucifer Yellow - pH_i, intercellular pH - PKC, protein kinase C - TPA, 12-0-tetradecanoylphorbol-13-acetate     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Morphological variations in gap junctions of ovarian granulosa cells.

Granulosa cells in ovarian follicles of rat, mouse, rabbit and hamster were studied by lanthanum tracer and freeze-fracture techniques. Abundant gap junctions exhibited striking intraspecific variation in size and pattern of particle aggregation. The smaller gap junctions showed close packing of the intramembranous A face particles. In large gap junctions, ranging up to 6 mu in diameter, particles were packed in rectilinear arrays separated by a labyrinthine network of particle-free 'aisles'. Small clusters of particles in a particle-poor circumferential zone suggested enlargement of junctions by peripheral accretion. Linear intramembranous structures, resembling those of occluding junctions, occasionally bounded large gap junctions. Spherical intracytoplasmic structures limited by gap junctional membranes were shown by tracer studies to arise by invagination of the cell surface. These were intrepreted as a means of disposal of junctions by interiorization.     

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system

Summary Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6–7 nm particles soley near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions) contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia.The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neural lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.     

The structural organization and protein composition of lens fiber junctions

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.     

Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development

During the 3-h developmental stage 14 in the chick, intercellular transfer of iontophoresed fluorescent dyes becomes less sensitive to the lowering of intracellular pH by either CO2 or acetate ions. Up to developmental state 14, dye transfer between lens cells is reversibly blocked by exposure to 50% CO2. Beyond stage 14, dye transfer between these cells is no longer reversibly blocked by elevated pCO2. Electronic coupling is present throughout lens development and is not reversibly blocked by high pCO2 at any stage. The gap junctions joining the lens cells show morphological changes at developmental stage 14. Up to stage 14, all gap junctions observed between chick lens cells have connexon assemblies that appear condensed or crystalline following routine freeze-fracture microscopy. Beyond stage 14, chick lens cells express gap junctions with both the condensed assemblies and the dispersed assemblies characteristic of adult lens gap-junction structure.     

Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures

The topological organization of the major rat liver gap junction protein has been examined in intact gap junctions and gap junction-derived single membrane structures. Two methods, low pH and urea at alkaline pH, were used to "transform" or "split" double membrane gap junctions into single membrane structures. Low pH treatment "transforms" rat liver gap junctions into small single membrane vesicles which have an altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after digestion with L-1-to-sylamido-2-phenylethylchloromethyl ketone-trypsin. Alkaline pH treatment in the presence of 8 M urea can split isolated rat liver gap junctions into single membrane sheets which have no detectable structural alteration or altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after proteolytic digestion, suggesting that these single membrane sheets may be useful for topological studies of the gap junction protein. Proteolytic digestion studies have been used to localize the carboxyl terminus of the molecule on the cytoplasmic surface of the intact gap junction. However, the amino terminus does not appear to be accessible to proteases or to interaction with an antibody that is specific for the amino-terminal region of the molecule in intact or split gap junctions. Binding of antibodies, that block junctional channel conductance, can be eliminated by proteolytic digestion of intact gap junctions, suggesting that all antigenic sites for these antibodies are located on the cytoplasmic surface of the intact gap junction. In addition, calmodulin gel overlays indicate that at least two calmodulin binding sites exist on the cytoplasmic surface of the junctional protein. The information generated from these studies has been used to develop a low resolution two-dimensional model for the organization of the major rat liver gap junctional protein in the junctional membrane.     

Gap junction morphology of retinal horizontal cells is sensitive to pH alterations in vitro

Summary Isolated goldfish retinae were incubated in NaHCO₃-reduced solutions, a treatment known to lower intracellular pH and to decrease gap-junction-mediated coupling between cells. The morphology of the gap junctions of horizontal cells examined by means of freezefracture replicas and ultrathin sections displays alterations after such treatment. The gap-junctional particles aggregate into dense clusters or crystalline arrays, whereas controls (pH 7.5) display a loose arrangement of particles. Incubation in NaHCO₃-reduced solution leads to the appearance, in ultrathin sections, of prominent, electron-dense material beneath the gap-junctional membranes. Both effects, the increasing density of particles and the appearance of electron-dense material, are reversible. The application of dopamine, which uncouples horizontal cells, and its antagonist haloperidol produce less clear-cut effects on particle density in vitro.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.