Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40-transformed human fibroblasts

Previous studies indicate that although normal and Simian virus (SV40)-transformed WI38 human fibroblasts have similar levels of intracellular Ca++ on a per mg protein basis, their ability to maintain this intracellular Ca++ against a low concentration of extracellular Ca++ differs markedly. The transformed but not the normal cells rapidly lose Ca++ when exposed to low extracellular Ca++, suggesting Ca++ transport and/or sequestration differ in the two cell types. In this study we have extended our investigations of Ca++ metabolism in the two cell types. We observe that normal WI38 cells, when exposed to metabolic inhibitors to deplete intracellular ATP, undergo a twofold increase in intracellular Ca++ levels. Under similar conditions and over the same time course, no comparable change in Ca++ level is observed in the SV40-transformed cell, despite the extensive depletion of ATP. 45Ca++ desaturation curves indicate that the bulk of the net increase in cell Ca++ following ATP depletion of the normal WI38 cell comes to reside in a slowly exchanging Ca++ pool. The data also indicate that glycolysis, and not oxidative phosphorylation, drives the active extrusion of Ca++ from these cells, an observation consistent with previous studies on the Na+-K+ pump in other cell types. Finally, the data indicate that in these cells mitochondria do not appear to be the major subcellular organelle responsible for regulation of at least the two cellular Ca++ pools measurable using isotope desaturation analysis. This is based on the inability of the respiratory inhibitor rotenone to alter significantly the size of either of these Ca++ pools. These pools compose 80-90% of total cell Ca++ in both cell types.     

Human bone cell cultures: a new model for studying the mechanism of action of calcitonin

An investigation on cell cultures obtained from temporal human bone fragments showed that they provide a suitable model for studying the mechanism involved in calcitonin action on bone cells. Furthermore they demonstrated: a transitory increase in 45Ca uptake that returned to control values ten minutes after the hormone was added; a relation between 45Ca uptake and increased cAMP concentrations when these were measured at the same time intervals; a reproduction of the salmon calcitonin (sCT) effect after incubation of the cultures with either db-cAMP or db-cGMP and inhibition of 45Ca uptake and parallel decrease in cAMP levels with propanol. These results suggest that in human bone cell cultures, sCT acts as a temporary promoter of 45Ca uptake, probably by activating an adenylate-cyclase system through a beta-receptor.     

The effects of lectins on the interaction between macrophages and bone in vitro

Summary Recent studies have demonstrated that the attachment of elicited rat macrophages to bone is mediated by specific saccharides located on the cell and/or bone surfaces. We have used a macrophage-bone culture system to study the effects of two lectins, concanavalin A (con A) and soybean agglutinin (SBA), on the morphology of macrophage attachment to a devitalized bone surface and subsequent functional activity. Macrophages were obtained from 3- to 4-week-old rats by peritoneal lavage and the adherent pool was used to prepare cell suspensions. Con A-treated, SBA-treated or control cell suspensions were aliquoted onto the endocranial surface of devitalized rat calvariae. The cells were allowed to attach for 1 h at 37° C, after which, the bone samples were removed from culture and prepared for scanning electron microscopy (SEM). The morphology of con A-treated macrophages attached to bone was markedly different from that of control or SBA-treated cells. Con A altered the attachment and subsequent spreading of macrophages on bone as visualized by SEM. Furthermore, the number of con A-treated cells that attached to bone and the average surface area of cell membrane apposed to the matrix was significantly different from that of control or SBA-treated cells. A ⁴⁵Ca bone-release assay was performed to evaluate the functional significance of the morphological findings. Lectin-treated or control cell suspensions were allowed to attach to the endocranial surface of ⁴⁵Ca pre-labeled calvariae for 1 h. Following attachment, the samples were cultured for 72 h. The con A-treated cultures demonstrated a significant decrease in the release of ⁴⁵Ca after 48 and 72 h in comparison to control cultures, while the ⁴⁵Ca released from SBA-treated cultures did not differ significantly from controls. These results suggest that certain sugar residues common to membrane-associated glycoconjugates and the organic component of the bone matrix regulate the attachment of macrophages to bone and their subsequent bone-resorbing activity.     

Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study

The objective of this study was to isolate fibrotic cells from human lung biopsies taken from different central pulmonary locations. A comparison was made of cell morphology, proteoglycan- and protein-expression in mesenchymal cell cultures obtained from human bronchial biopsies from patients with asthmatic-like disorders. We isolated viable cells from 10 out of the 12 biopsies. The fibroblast-like cells were positive for the biomarker a-smooth muscle actin, indicating that the cells were in an activated state. Two different types of fibroblast-like cells were observed from human pulmonary connective tissue; one of contractile type with lamellipodia that facilitate migration and a second cell type with an increased cell size, which most likely is of a synthetic phenotype. This is the first evidence of alterations in the proteoglycan expression pattern of versican, perlecan, biglycan and decorin which can be linked to the pathophysiological state of asthmatics proven by a combination of solid-phase extraction by reversed phase and by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein expression analysis using two-dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity within biopsies derived from different locations of the same patient.     

Proliferation of human embryonic and fetal epidermal cells in organ culture

The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.     

Extracellular products of Staphylococcus aureus reversibly inhibit the terminal differentiation of cultured mouse epidermal cells

The effect of extracellular products from Staphylococcus aureus on the differentiation of mouse epidermal cells was studied using an in vitro cell culture system. The extracellular products from a clinical strain of S. aureus isolated from human skin lesions reversibly inhibited the Ca++-induced terminal differentiation of epidermal cells, as determined by their morphology and the extent of cornified envelope formation. This suggests that a similar modification of cell differentiation is involved in the pathogenesis of S. aureus-induced skin disease.     

Heterogenecity of stromal cell precursers isolated from rat bone marrow

Bone marrow stroma contains mesenchymal stem cells (MSC) which are progenitor cells, at least for tissues arising from mesechyma. The study of MSC biology yields controversial data. Therefore further experiments are needed to characterize these cells. The aim of our research was to compare primary cultures and subcultures of stromal precursor cells isolated from rat bone marrow. Long-term cultures of these cells isolated from 5 animals have been obtained. Morphological, immunophenotypic, and functional (capacity to osteogenic differentiation) characteristics of the cells have been investigated. We show that the cell morphology in the cultures is highly heterogenic. Morphological cell types are described. Heterogeneity of stromal cells declines on late passages. Cell cultures isolated from different animals have the same immunophenotypic markers (CD90, CD44, CD54, CD106, CD45, CD11b) but different morphological characteristics and a different capacity to osteogenic differentiation during long-term cultivation. The data show that more specific markers and functional tests should be applied to identify MSC.     

Cell cycle distribution of primitive haematopoietic cells stimulated in vitro and in vivo

A novel approach is used to study the proliferating behaviour of primitive haematopoietic cell populations in response to different stimuli. A mathematical model based on the average proportion of apoptotic, dividing and quiescent cells in primitive haematopoietic cell populations is developed to describe the mitotic history of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester-labelled cells. The cell cycle distributions in different cytokine-supplemented cultures of primitive human and mouse bone marrow cells are determined and compared with those found in vivo. The results indicate that a combination of flt-3 ligand, Steel factor and interleukin-11 or hyper-interleukin-6 provide a level of mitogenic stimulation similar to that existing in vivo after a myeloablative radiation dose. The comparison of the cell cycle distribution obtained for different cultures of human bone marrow CD34(+)(45RA/71)(-) cells demonstrates that the addition of flt-3 ligand in these cultures decreases apoptosis significantly but does not reduce quiescence. In addition, in vivo and in vitro, it was found that more than 3 days of stimulation are required to recruit a maximum number of quiescent cells into active cell cycle. These kinetics of cell cycle activation are found to be similar to those identified for the haematopoietic stem cells compartment in the same cultures. This mathematical analysis provides a useful tool for the development of haematopoietic stem cell culture processes for clinical applications.     

IgE-mediated release of histamine from human cutaneous mast cells

We investigated the ability of antigen-IgE interactions to stimulate histamine release from human infant cutaneous mast cells. Skin obtained at circumcision contained numerous perivascular mast cells, as assessed by light and electron microscopy. The histamine content of this tissue averaged 17.7 ng (+/- 1.5 SEM)/mg wet weight. Challenge of 200-microns thick sections of unsensitized skin with varying concentrations of monoclonal murine antibodies to human IgE caused no net release of histamine. After skin sections were incubated in the presence of 5 micrograms/ml of human myeloma IgE (S) for 120 min at 37 degrees C, monoclonal anti-IgE challenge resulted in 40.1% (+/- 6.0 SEM) histamine release. Similar passive sensitization with 1/20 dilutions of serum from humans expressing IgE to purified Juniperus sabinoides (JS) antigen rendered the tissue responsive to specific antigen challenge. Dose-related histamine release occurred over 30 min with optimal release of 12.6% (+/- 2.4 SEM) after stimulation with 100 ng/ml of JS antigen. This reaction required sensitization with serum containing IgE to JS and was antigen-specific. Optimal reactions to antigen occurred at 3 mM added Ca++, 34 degrees C to 37 degrees C, pH 7.2. Antigen-induced release was markedly influenced by the added Ca++ concentration; no release occurred in the absence of Ca++, 54% of the optimal response was observed at 2 mM Ca++, and 28% of the optimal response occurred at 4 mM Ca++. The addition of Mg++ did not influence antigen-induced release. The results of this study provide functional evidence that 1) human infant cutaneous mast cells express Fc-epsilon receptors; 2) these receptors are largely unoccupied in vivo; and 3) stimulation of passively sensitized infant mast cells with anti-IgE or specific antigen leads to immediate histamine release. This new system should permit detailed in vitro studies of immediate hypersensitivity reactions in human skin.     

Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca++ and antagonism by adenosine 3'':5''-monophosphate

The plant lectin, concanavalin A (Con-A), and the ionophore, A-23187 (specific for divalent cations), stimulated glucose transport in rat thymocytes. Con-A stimulation developed more slowly and was somewhat less extensive than that of stimulation developed more slowly and was somewhat less extensive than that of A-23187. Both responses showed saturation dose dependencies. The two responses were poorly additive, suggesting that A-23187 may saturate regulatory processes shared by the two stimulatory mechanisms. Doses of methylisobutylxanthine (MIX) and prostaglandin E2 which raised adenosine 3':5'-monophosphate (cAMP) levels in these cells also antagonized the Con-A stimulation of glucose transport but did not inhibit basal glucose transport or the A-23187 stimulation. Dibutyryl-cAMP and 8-bromo-cAMP also natagonized Con-A stimulation without inhibiting basal glucose transport. MIX antagonized high Con-A doses about as strongly as it did low Con-A doses, suggesting that MIX did not compete in the Con-A binding step or other process saturable by Con-A. [3H-A1Con-A binding was not affected by MIX. The stimulatory effects of Con-A and A-23187 were reduced by reduction of Ca++ in the medium. Both Con-A and A-23187 enhanced 45Ca++ influx and cellular Ca++ content. The A-23187 dose, which was saturating for glucose transport stimulation, enhanced Ca++ influx and cellular Ca++ content more than did the Con-A dose which was saturating for glucose transport stimulation. The dose fo MIX which specifically antagonized Con-A stimulation of glucose transport proved also to reduce Ca++ influx and cellular Ca++ in the presence of Con-A but not in the presence of A-23187. Thus, glucose transport correlates rather well with cellular Ca++. These results are compatible with the view that Ca++ in a cellular compartment can promote glucose transport, the Con-A's enhancement of Ca++ entry contributes to its stimulation of glucose transport, and the MIX antagonized Con-A action at least partly by reducing Ca++ entry. The action of MIX is apparently mediated by cAMP.     

Comparison of human interleukin-1 beta and its 163-171 peptide in bone resorption and the immune response

Human interleukin-1 beta (IL-1 beta) caused a dose- and time-dependent enhancement of the release of 45Ca from prelabeled mouse calvaria in organ culture. In addition, IL-1 beta dose-dependently stimulated the formation of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha in the calvarial bones. However, IL-1 beta-induced 45Ca release was only partially inhibited by blocking the PGE2 response with indomethacin, suggesting that enhanced PGE2 formation in response to IL-1 beta is not necessary to obtain a bone resorptive effect, but that prostaglandins potentiate the action of IL-1 beta. The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, administered simultaneously with antigen (SRBC) to C3H/HeN male mice, induced a dose-dependent enhancement of specific antibody-producing cells in the spleen (PFC). The degree of PFC stimulation was comparable to that caused by native human IL-1 beta. In mouse bone cultures, neither 45Ca release nor prostanoid formation was stimulated by fragment 163-171. These data indicate that (1) IL-1 beta-induced stimulation of bone resorption is dissociable from IL-1 beta-induced increase of prostanoid biosynthesis and (2) the epitope of the IL-1 beta molecule involved in the immunostimulatory effects may be different from that involved in the stimulatory effects on bone resorption.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min). The heat-induced increase in 45Ca++ influx was linearly related to membrane accumulation of phosphatidic acid, phosphoinositide metabolite that may be involved in Ca++ gating. These results suggest that the membrane may be the proximal target of heat shock; stimulation of rapid breakdown of polyphosphoinositides and subsequent increases in intracellular free Ca++ may provide a mechanistic insight into the pleiotropic effects of heat. In addition, the large increases in Ca++ influx could initiate a Ca++ dependent mechanism of thermal cell killing.     

Retinoic acid suppresses interleukin 6 production in normal human osteoblasts

Systemic long-term retinoid therapy for chronic skin diseases significantly reduced bone turnover markers within days and led to bone abnormalities. Retinoic acid (RA) plays a key role in the regulation of mouse bone cell proliferation, differentiation and functions. Meanwhile, there is little information of RA effect on human osteoblast and osteoclast cell development and function. Interleukin 6 (IL-6) is a pleiotropic cytokine with profound effects on bone metabolism. Thus, the present study examined the RA effect on cell differentiation, alkaline phosphatase and osteocalcin production as well as IL-6 production in normal human osteoblasts. The number of large differentiated osteoblast cells decreased in RA-treated cultures P<0.05. The production of bone specific markers, alkaline phosphatase and osteocalcin, was also reduced in RA-treated cultures. Normal human osteoblasts produced 31.0+/-4.8 pg IL-6 per ml in control cultures. Within 24 h, RA at all four concentrations reduced Il-6 production from normal human osteoblasts. The pharmacological concentration of 10(-5) M RA suppressed 90% of IL-6 production. The present study shows for the first time that RA profoundly inhibits IL-6 production in normal human osteoblasts within 24 h and in a dose-dependent manner. RA was shown previously to inhibit IL-6 production in several other normal and malignant human cell types. The associated decrease in osteoblast cell differentiation, alkaline phosphatase and osteocalcin production could result from the rapid RA-inhibition of IL-6 production. Thus, RA inhibition of IL-6 production in normal human osteoblasts may contribute to the bone abnormalities seen after systemic long-term retinoid therapy in some patients.     

Functional and biochemical characterization of osteoclast-like cells derived from giant cell tumours of bone.

Cells harvested from human giant cell tumours of bone were characterized on the basis of morphological features, proliferative capacity, total(AP) and tartrate resistant acid phosphatase (TRAP) activity, and hormonal response. Culture were formed by mononucleated and multinucleated cells. Mononucleated cells showed fibroblastic morphology, whereas multinucleated cells showed osteoclastic phenotype. We conclude that in these cultures mature osteoclasts and their mononuclear precursors are present.     

In vitro analysis of proliferating epithelial cell populations from the mouse mammary gland: Fibroblast-free growth and serial passage

Summary Normal and neoplastic mouse mammary epithelial cells were cultured in nutrient medium containing D-valine substituted for L-valine. Fibroblast overgrowth was prevented and epithelial cell functions and morphology were retained in cultures maintained in, D-valine medium up to 2 months. A nonenzymatic technique was devised to dissociate epithelial cell monolayers. The combined use of this dissociation buffer and D-valine nutrient medium made it possible to passage serially normal and neoplastic mammary epithelial cells. Normal cells were derived from mammary glands of animals stimulated with exogenous hormones for various periods. The period of in vivo hormonal stimulation influenced the ability of normal mammary epithelial cells to attach and proliferate in primary and serially passaged cultures. A greater proportion of cells derived from glands following 2 to 4 weeks of hormonal stimulation were recovered after replating and showed higher labeling indices during serial passage than cells from unstimulated or 5- to 7-week stimulated groups. This investigation was supported by Grant No. CA 05388 from the National Cancer Institute and by Cancer Research Funds of the University of California.     

Multi-lineage potential of human mesenchymal stem cells following clonal expansion

Bone marrow contains mesenchymal cells that can be isolated and grown in vitro. Using appropriate treatment protocols such cultures can be induced to differentiate to yield osteoblasts, adipocytes, and chondrocytes. However, previous experiments had not addressed the question whether single pluripotent stem cells exist and can give rise to these different cell lineages or whether bone marrow mesenchymal cell preparations represent a mixture of committed precursors. We have used human adult bone marrow-derived mesenchymal cells obtained from iliac crest biopsies to demonstrate clonal outgrowth after limiting dilution and we show that some clones can be expanded over more than 20 cumulative population doublings and differentiated to osteoblasts, adipocytes, and chondrocytes. Our data provide direct experimental evidence that cultures of bone marrow-derived mesenchymal cells contain individual cells that fulfil two essential stem cell criteria: (i) extensive self-renewal capacity and (ii) multi-lineage potential.     

Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function: Implications for Off-The Shelf ES-MSC Therapies

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.     

Identification of cells from fetal bladder epithelium in human amniotic fluid

Summary Cultured human amniotic fluid cells consist of five different types of cytokeratin-positive epithelial cells, E-1 to E-5, differing by their size, growth morphology, and cytokeratin pattern, according to our earlier investigations. Using anticytokeratin antibodies in indirect immunofluorescence (IIF) microscopy, we show in this study that cultured urine cells contain four of the cell types found in amniotic fluid. In addition, we used two urothelium-specific antibodies, anti-UMA and anti-Las-86, in combination with cytokeratin antibodies to distinguish urothelium-derived cells in amniotic fluid and urine cell cultures. Two of the epithelial cell types were found to express urothelial antigens and thus to originate from the transitional bladder epithelium. These cells were found in 26 of the 33 amniotic fluid cell cultures and in nine of the ten urine cell cultures.     

EGTA induces prolonged summed depolarizations in Mytilus gill coupled ciliated epithelial cells: implications for the control of ciliary motility

Abfrontal ciliated cells of Mytilus edulis gill beat when mechanically stimulated, a consequence of a Ca++-based generator potential and regenerative response. In contrast, the lateral ciliated epithelial cells arrest when stimulated, a consequence of a Ca++-based generator potential and a Na+/Ca++-based regenerative response. Iontophoretic injection of EGTA in abfrontal cells, followed by mechanical stimulation, results in a large, prolonged depolarization that returns to the resting level stepwise. It has been hypothesized that this phenomenon is caused by successive Ca++-dependent repolarizations in coupled cells, first in adjacent cells and then in the injected cell, in accord with relative EGTA loading. We have now demonstrated this same stepwise repolarization phenomenon in the Na+/Ca++-dependent lateral ciliated cells. In this case, each repolarization step is often preceded by a small spike. With either cell type, using two-electrode recording techniques, we can detect the stepwise repolarization in distant cells, proportionately decremented when the second (KCl) electrode is some distance from the injection (EGTA) electrode and stimulus. When force is applied between the electrodes and nearest the KCl electrode, a greater initial response is recorded from this electrode, presumably resulting from depolarization of its impaled cell, prolonged by EGTA diffusion through the intervening cell junctions. The subsequent repolarization steps are of approximately the same size, suggesting repolarization of cells between the two electrodes. These observations are consistent with the cell coupling/EGTA loading hypothesis and indicate that both cell types mediate repolarization through Ca++ and propagate ciliary beat or arrest through intracellular coupling.