Cinemicrographic observations of cultured adrenocortical tumor cells. Dynamic responses to ACTH and cytochalasin B

ACTH increases the basal steroidogenic activity of cultured adrenocortical tumor cells, whereas moderate-high doses of cytochalasin B (CB) inhibit both basal and ACTH-induced steroidogenesis. Previous ultrastructural studies have revealed that ACTH rearranges microfilaments in these adrenal cells, whereas CB causes microfilaments to aggregate into felt-like masses. It has been postulated that the ACTH effects may facilitate organelle motility and increase organelle interactions that are required for steroid biosynthesis, and that the CB-created "foci" may impede or prevent the organelle meetings. To shed light on these possibilities, we have employed 16 mm cinemicrography of unstimulated adrenal tumor cells and cells incubated for 1-2 h with ACTH (10 mU/ml), or low (10 micrograms/ml), or high (50 micrograms/ml) doses of CB. ACTH caused initial increases in membrane ruffling and a "flurry" of particle (organelle) activity above that seen in unstimulated cells. The stimulated cells then retracted from each other and began their characteristic "rounding up" in response to the hormone. Particles appeared to move towards the nucleus, and in fully-rounded cells were extremely congested. Steroid production rose several fold above basal levels. CB10 produced slight-marked cell convexities, nearly stopped particle motility and inhibited steroid production moderately. CB50 produced an asymmetrical, spidery cell form, stopped membrane ruffling and particle motility and abolished steroidogenesis. After a washout of CB50, particle motility resumed nearly immediately. Our CB data indicate that associations between particles, presumably between mitochondria and various sources of cholesterol, are prerequisite for basal steroidogenesis in the adrenal tumor cells. In ACTH-stimulated cells, increases in steroid output correspond with increased opportunities for particle associations. These opportunities appear to arise directly or indirectly from ACTH effects on microfilaments. The responses of microfilaments to the hormone may be particularly intense in tumorous forms. By these means, the cells may express their differentiated function, although their cytoplasm has a distinctly unspecialized appearance.     

Dibutyryl cyclic AMP modulation of gap junctions in SW-13 human adrenal cortical tumor cells

Cultured human adrenal cortical adenocarcinoma cells (SW-13) form a confluent monolayer of epithelial-like cells when seeded into culture flasks. Following a 24-48 hr non-mitotic period, cells begin to divide and become confluent within a week after seeding at 5 X 10(4) cells/cm2. The SW-13 cells were exposed to dibutyryl cyclic AMP (DbcAMP), cyclic AMP (cAMP), sodium butyrate, and adrenocorticotropin (ACTH). The rate of SW-13 cell proliferation was measured with a DNA microfluorometric assay, as well as by procedures measuring the incorporation of 3H-thymidine. In addition, following administration of ACTH and DbcAMP, the fractional area of membrane covered by gap junctions was quantitated with freeze-fracture electron microscopic techniques. Dibutyryl cyclic AMP at a concentration of 1 X 10(-3) M decreased the growth rate of the cell population. There was a corresponding increase in the fractional area of gap junctions found on the cell membrane in 96-hr DbcAMP-treated cultures. ACTH (40 mU/ml) exposure failed to produce an increase in the fractional area of gap junctions or to alter the rate of cell proliferation. From these data it can be suggested that elevations in cAMP levels within the cell can be related to both the proliferation of gap junctions and the decrease in cell proliferation in the SW-13 tumor cell.     

Hormone-induced intercellular signal transfer dissociates cyclic AMP- dependent protein kinase

We used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact-dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and occurs in the absence of apparent cell fusion. The only structural feature connecting Y-1 and granulosa cells were gap junctions implying they provided the communication channels; however, alternative mechanisms cannot be excluded. We have not established the identity of the signal being transferred although cAMP is a logical candidate.     

Hormonal regulation of gap junction differentiation

Thin-section, tracer, and freeze-cleave experiments on hypophysectomized Rana pipiens larvae reveal that gap junctions form between differentiating ependymoglial cells in response to thyroid hormone. These junctions assemble in large particle-free areas of the plasma membrane known as formation plaques. Between 20 and 40 h after hormone application, formation plaque area increases approximately 26-fold while gap junction area rises about 20-fold. The differentiation of these junctions requires the synthesis of new protein and probably RNA as well. On the basis of inhibitor experiments, it can be reported that formation plaques develop at about 16-20 h after hormone treatment and stages in the construction of gap junctions appear 4-8 h later. These studies suggest that gap junction subunits are synthesized and inserted into formation plaque membrane during the differentiation of the anuran ependymoglial cells.     

A DISTINCTIVE CELL CONTACT IN THE RAT ADRENAL CORTEX

Extensive cell contacts which resemble septate junctions occur between cells in the three major zones of the rat adrenal cortex. Characteristically, they extend between small intercellular canaliculi and the periendothelial space, frequently interrupted by gap junctions and rarely by desmosomes. Zonulae occludentes have not been identified in the adrenal cortex. Along this distinctive cell contact, the cell membranes of apposing cells are separated by 210–300 a bisected by irregularly spaced 100–150-A extracellular particles which are often circular in profile. In lanthanum preparations, these particles appear to form a continuous chain throughout the intercellular space and are visualized as an alveolate structure in sections parallel to the plane of the cell membrane. The cell membrane in the area of septate-like contact does not differ from nonjunctional areas of the cell membrane in freeze-fracture replicas. The cell contact retains its integrity after cell dispersion and after the separation of cell membranes from disrupted cells. The intercellular particles also persist after brief extraction in lipid solvents. Besides adherence, possible functions of this adrenal contact include maintenance of the width of the extracellular space, the provision of channels between intercellular canaliculi and the bloodstream, and utilization as cation depots. Similar structures are also present between adrenal cortical cells of several other species and between interstitial cells of the testis. This type of cell contact may, in fact, be a typical feature of steroid-hormone-secreting tissues in vertebrates.     

Adrenocortical atrophy of hypophysectomized rats can be reduced by corticotropin-releasing hormone (CRH)

Summary The effects of high dose injections of corticotropin-releasing hormone (CRH) on the adrenal cortex of hypophysectomized rats were studied at the light-and electron-microscopical levels. Adrenocortical atrophy induced by hypophysectomy could be reduced by daily i.p. injection of 10 g (3 nmol) CRH given for 3 days starting at day 5 after the operation. The cortex broadened, mostly because of hypertrophy of the zona fasciculata. Blood vessels were enlarged. Although the adrenocortical cells of hypophysectomized rats showed features of a functionally suppressed state, such as tubular mitochondria, the cells of CRH-treated animals showed characteristics of stimulated cells. The inner membrane of the mitochondria formed the typical densely packed vesicles of adrenocortical cells that are active in steroidogenesis. Lipid droplets were found to be reduced, and the cells developed filopodia at their surface. These morphological observations indicate that CRH influences the adrenal cortex via extrapituitary mechanisms.     

VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells.     

Redistribution of surface macromolecules in dissociated epithelial cells

A number of ultrastructural and cytochemical techniques were used to study intact epithelial cells lining the frog urinary bladder: high resolution autoradiography after administration of [3H]glucosamine or [3H]fucose; 125I iodination of external protein; concanavalin A-peroxidase, periodic acid-chromic acid silver methenamine; and colloidal thorium. Results indicate that the material (probably glycoprotein) coating the apical surface differs from that which lines the lateral and basal surfaces. After dissociation and isolation of the epithelial cells, the material previously confined to the apical surface invaded progressively the opened "tight junctions" (about 5 min), then the lateral membranes (about 40 min), and finally the basal membrane (about 80 min): at that time, the whole cell surface was entirely enveloped by the apical material. Since, on the one hand, the reacting material was confined to the apical surface when the tight junctions were closed (in intact epithelial cells) and since, on the other hand, the apical material was sliding down the laterobasal membranes when the tight junctions were opened (in dissociated cells), it may be concluded that tight junctions contribute to maintain the cell surface specialization in epithelia.     

Formaldehyde-induced appearance of septate junctions between digestive vacuoles

Tissue biopsies from (1) some chronic inflammatory diseases, (2) a necrotic tumoral process, (3) normal human lymphatic ganglia, and (4) two congenital diseases of the adrenal cortex were selected for study. A block from each biopsy was fixed in glutaraldehyde-paraformaldehyde; a second block was fixed in 10% formaldehyde. In all cases septate junctions between digestive vacuoles did occur in phagocytic cells and some adrenal cortex cells fixed in formaldehyde. These junctions were similar to those reported recently for malakoplakia phagocytes. Consistently, they were not found to attach organelles other than lysosomes derivatives. Both phagocytes and adrenal cortex cells in the material fixed in glutaraldehyde-paraformaldehyde did not display adhesive specializations between digestive vacuoles. This suggests that the septate junctions described herein are artifactuous structures induced by formaldehyde. There is, however, a certain degree of specificity of cells having the capability of developing these septate junctions. It is assumed that the coating material of digestive organelles in phogocytes and some other cells would be responsible for both cell specificity and organelle specificity of the formaldehyde-induced septate junctions.     

Organization of the nematocyst battery in the tentacle of hydra: Arrangement of the complex anchoring junctions between nematocytes,epithelial cells,and basement membrane

Summary Nematocytes (stinging cells) of hydra tentacles are anchored to the basement membrane by peculiar complex junctions in which a flattened tongue of an epithelial cell is interposed between the nematocyte and the basement membrane. In this paper we describe the arrangement of these junctions with emphasis on how they are related to the architecture of the epithelial cell. Each epithelial cell, called a battery cell, harbors 10–20 nematocytes and bears muscle processes that extend along the basement membrane. The epithelial cell component of the complex junction is usually a lateral extension of a muscle process. All nematocytes within a battery cell make junctions with muscle processes of the same (resident) epithelial battery cell despite the presence of numerous muscle processes from adjacent (foreign) cells. Some nematocytes make junctions with several resident processes, spanning the foreign processes to do so. Most junctions reside near the proximal ends of the muscle processes. New findings are reported on the substructure of the junctions. They are composed of aggregates of smaller elements, and the cytoskeleton within the complexes has a pronounced longitudinal organization. These observations are consistent with a suggestion that the complex junctions develop by aggregation of smaller junctional units originating elsewhere on the cells.     

Hormonal modulation of ovarian interstitial cells with particular reference to gap junctions

Thin-section and freeze-fracture studies on the rat ovarian interstitial cells revealed reductions in the size and the number of gap junctions after pituitary ablation. Small gap junctions, however, persist as long as 90 days after hypophysectomy even though regressive cytoplasmic changes are completed 75 d earlier. Administration of exogenous human chorionic gonadotrophin (HCG) results in the restoration of the normal interstitial cell morphology which is accompanied by amplification of junctional membrane. Within 24 h of hormone application, gap junction growth is characterized by the appearance of formation plaques. These studies suggest that the effect of hormone on interstitial cell gap junctions is to modulate the junctional surface area.     

Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

Intercellular communication and tissue growth: IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells

Summary The structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of gap junction; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related. The junctions of the intermediate hybrid cells with incomplete channel-competence (characterized by cell-to-cell transfer of small inorganic ions but not of fluorescein), present special intramembranous fibrillar structures instead of discrete gap-junctional particles. The possibility that these structures may constitute coupling elements with subnormal permeability is discussed in terms of incomplete dominance of the genetic determinants of gap junction.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).     

Revisiting the Stimulus-Secretion Coupling in the Adrenal Medulla: Role of Gap Junction-Mediated Intercellular Communication

The current view of stimulation-secretion coupling in adrenal neuroendocrine chromaffin cells holds that catecholamines are released upon transsynaptic sympathetic stimulation mediated by acetylcholine released from the splanchnic nerve terminals. However, this traditional vertical scheme would merit to be revisited in the light of recent data. Although electrical discharges invading the splanchnic nerve endings are the major physiological stimulus to trigger catecholamine release in vivo, growing evidence indicates that intercellular chromaffin cell communication mediated by gap junctions represents an additional route by which biological signals (electrical activity, changes in intracellular Ca²⁺ concentration,…) propagate between adjacent cells and trigger subsequent catecholamine exocytosis. Accordingly, it has been proposed that gap junctional communication efficiently helps synapses to lead chromaffin cell function and, in particular, hormone secretion. The experimental clues supporting this hypothesis are presented and discussed with regards to both interaction with the excitatory cholinergic synaptic transmission and physiopathology of the adrenal medulla.     

Differential redistribution of protein kinase C in human aldosteronoma cells and adjacent adrenal cells stimulated with ACTH and angiotensin II

We have examined protein kinase C activity and hormone secretion in aldosteronoma cells derived from adrenocortical glomerulosa cells and in adjacent adrenal cells containing adrenocortical fasciculata-reticularis cells. When aldosteronoma cells were stimulated with ACTH or angiotensin II, protein kinase C activity gradually decreased in cytosol whereas it increased in membrane. Coincident with the changes of protein kinase C activity, there was enhancement of secretion of aldosterone. On the other hand, incubation of adjacent adrenal fasciculata-reticularis cells with ACTH induced cortisol secretion and an increase in cytosolic protein kinase C activity, accompanied by a decrease in the enzyme activity in membrane. Upon stimulation with angiotensin II, adjacent adrenal fasciculata-reticularis cells did not secrete cortisol and no significant changes of protein kinase C activities were observed in either cytosolic or membrane fractions. These results indicate that both ACTH and angiotensin II stimulate aldosterone secretion and cause translocation of protein kinase C from cytosol to membranes in aldosteronoma cells, whereas, in fasciculata-reticularis cells, only ACTH stimulates cortisol secretion and this is associated with translocation of protein kinase C in the opposite direction, viz., from membrane to cytosol.     

The role of the cytoskeleton in the regulation of steroidogenesis

The slow step in steroid synthesis involves the transport of cholesterol from lipid droplets in the cytoplasm to the first enzyme in the pathway—the cytochrome P450 that converts cholesterol to pregnenolone (P450scc) which is located in the inner mitochondrial membrane. ACTH stimulates this intracellular transport of cholesterol in adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells) and this effect of the trophic hormone is inhibited by cytochalasins, by anti-actin antibodies and DNase I suggesting that the response to ACTH requires a pool of monomeric (G-) actin that can be polymerized to F-actin. Recent studies have shown that lipid droplets and mitochondria of adrenal cells are both attached to intermediate filaments. Moreover ACTH reorganizes the cytoskeleton and changes the shape of the cell. These observations suggest a mechanism for transport of cholesterol that involves reorganization and contraction of actin microfilaments which may, in turn, cause movement of droplets and mitochondria together through their common attachment to intermediate filaments.     

Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

The dynamic control of E-cadherin is critical for establishing and maintaining cell-cell junctions in epithelial cells. The concentration of E-cadherin molecules at adherens junctions (AJs) is regulated by lateral movement of E-cadherin within the plasma membrane and endocytosis. Here we set out to study the interplay between these processes and their contribution to E-cadherin dynamics. Using photoactivation (PA) and fluorescence recovery after photobleaching (FRAP), we were able to monitor the fate of E-cadherin molecules within the plasma membrane. Our results suggest that the motility of E-cadherin within and away from the cell surface are not exclusive or independent mechanisms and there is a fine balance between the two which, when perturbed, can have dramatic effects on the regulation of AJs.Key words: E-cadherin, adherens junctions, plasma membrane, protein dynamics, photoactivation, photobleaching