Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.

Rod and cone degeneration in the rd mouse is p53 independent.

PURPOSE: To determine whether p53 is required for the death of rod and cone photoreceptors in rd mice, a model of human retinitis pigmentosa, and/or for the natural degeneration of inner nuclear layer (INL) cells in the developing retina. METHODS: Rod photoreceptor and INL apoptosis was assessed by TUNEL staining of mouse sagittal sections from post natal day (P) 10, 13, 15, 17, and 20 day p53+/+ and p53-/- rd retinas. Cone photoreceptor survival was measured by counting the total number of peanut agglutinin (PNA) positive cells in eighty four 0.25 mm x 0.25 mm bins in each eye, distributed equally across the four quadrants of whole mount retinas from 3 month old p53+/+ and p53-/- rd retinas. RESULTS: Both the kinetics of rod and INL cell death as well as the survival of cones were essentially unaffected by the absence of p53. CONCLUSIONS: Despite established links with retinal apoptosis, p53 is not essential for rod or cone cell degeneration in the rd mouse or for the elimination of bipolar and Muller cells during late retinal development.

Adhesion of transplanted chondrocytes onto cartilage in vitro and in vivo.

OBJECTIVE: The specific objectives of this study using organ culture were (1) to transplant chondrocytes onto an intact cartilage surface; (2) to genetically modify endogenous and transplanted chondrocytes; and (3) to assess the ability of these cells to continually express a gene product. The specific objective with in vivo experiments was to transplant chondrocytes with intraarticular injections to cartilage. METHODS: Fluorescent membrane and intracellular dyes were used in conjunction with confocal microscopy to observe the integration of transplanted chondrocytes into cartilage both in vitro and in vivo. The distribution and duration of binding of rat, canine, and bovine chondrocytes to cartilage explants and the duration of expression of genes transduced into the transplanted chondrocytes were also determined. We used the vector AdlacZ, an E1 and E3 deleted replication defective adenoviral vector that contains the beta-galactosidase gene driven by the beta-actin promoter and the cytomegalovirus enhancer. RESULTS: The transplanted chondrocytes had a patchy distribution after in vitro or in vivo transplantation and buried themselves within the cartilage over time. Chondrocytes infected with the adenoviral vector AdlacZ soon or well after transplant to cartilage explants were maintained on the cartilage and continued throughout the duration of each trial to produce beta-galactosidase coded by the adenoviral vector. The cartilage plugs were infected with AdlacZ at 2 days or one, 2, 5, or 8 weeks after the chondrocytes were transplanted. The cartilage slices were then cultured from 15 days for chondrocytes infected at 8 weeks to 60 days for chondrocytes infected at 2 days post-transplant before determining the expression of beta-galactosidase. CONCLUSION: These results support the possibility of repairing cartilage by intraarticular injections of chondrocytes. Transduction of chondrocytes with genes producing a variety of matrix promoting proteins should further enhance the reconstruction of osteoarthritic cartilage.

Factors affecting the efficacy of bovine chondrocyte transplantation in vitro.

Current therapies for osteoarthritis have been primarily directed at symptom relief rather than disease modification or cure. Improved understanding of cartilage biology and metabolism has permitted exploration of disease-modifying treatments for OA. Chondrocyte transplantation is one approach to disease modification that has received increasing attention. To date, most chondrocyte transplantation has focused on surgical implantation into isolated chondral defects.Our hypothesis is that cultured chondrocytes will preferentially transplant to hyaline cartilage after intraarticular injection. The purpose of this study was to quantify chondrocyte adherence to cartilage in an in-vitro bovine explant model under differing culture conditions. The effect on chondrocyte transplantation of time, of alginate vs. monolayer culture techniques, and of differing origin of tissue explants within the knee joint were assessed. The effect on transplantation of physically modifying the explant surface was also assessed. In addition to quantification of transplantation adherence, the morphology of transplanted chondrocytes was assessed with confocal and electron microscopy. Maximal adherence occurred by 24 h post-transplantation. Baseline transplant densities exceeding 1 x 10(6) cells/cm(2)were observed on unmodified cartilage surfaces. No significant differences in binding density were noted between cartilage explants obtained from the patella, femoral condyles, tibial plateaus or the trochlear groove. In addition, no differences in chondrocyte adherence were noted in cells cultured in monolayer or alginate beads. Transplanted chondrocytes were noted to be spherical irrespective of the culture methods employed. Notably, chondrocytes demonstrated significantly improved adherence to cartilage surfaces after the superficial layer was removed as compared to normal intact cartilage surfaces (increase of 26%, P< 0. 01). This suggests that chondrocytes may preferentially adhere to cartilage surfaces where the superficial layer has been damaged, as is the case in isolated chondral lesions, or with diffuse cartilage degeneration.

Resistance of the dopamine D2L receptor to desensitization accompanies the up-regulation of receptors on to the surface of Sf9 cells.

Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.

Agonist-induced desensitization of dopamine D1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from D1 receptor internalization.

The regulation of the dopamine D1 receptor was investigated by using c-myc epitope-tagged D1 receptors expressed in Sf9 (fall armyworm ovary) cells. Treatment of D1 receptors with 10 microM dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. After 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% of the cell surface receptors were internalized into an intracellular light vesicular membrane fraction as determined by radioligand binding and supported by photoaffinity labeling, immunocytochemical staining, and immunoblot analysis. Pretreatment of cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization, indicating a biochemical separation of these processes. Collectively, these findings indicate that the desensitization of D1 receptor-coupled adenylyl cyclase activity and D1 receptor internalization are temporarily and biochemically distinct mechanisms regulating D1 receptor function following agonist activation.

Dopamine D1 receptor distribution in Sf9 cells imaged by confocal microscopy: a quantitative evaluation.

A c-myc epitope-tagged human dopamine D1 receptor (c-myc D1 receptor) was expressed in Sf9 cells and its cellular distribution under basal conditions and after exposure to the agonist dopamine was examined. In the basal state, immunofluorescently labeled c-myc D1 receptors imaged by confocal microscopy appeared as a bright ring of label predominantly on the cell surface, and to a lesser extent as intracellular clusters of label. This pattern of receptor distribution was confirmed by radioligand-binding assays on plasma membrane and light membrane fractions using the D1 receptor-antagonist [3H]-SCH-23390. After exposure to dopamine, c-myc D1 receptors were redistributed on the cell surface, changing from a continuous ring to a discontinuous pattern of label. Analysis of fluorescence intensity and three-dimensional computer reconstruction of labeled receptors revealed a 30% decrease in surface labeling with no decrease in total number of receptors confirmed by radioligand-binding analysis. These findings constituted the first direct evidence of agonist-induced D1 receptor internalization. The results showed that the combination of confocal microscopy and three-dimensional reconstruction can be used to visualize and assess receptor distribution in Sf9 cells.

Inhibitory effects of alpha-interferon on epidermal growth factor-mediated receptor-dependent events.

To examine the mechanisms by which alpha-interferon (IFN-alpha) inhibits growth factor-mediated proliferative responses, we examined specific ligand-activated, receptor-dependent events. In direct ligand binding studies, we showed that IFN-alpha treatment of cells leads to a reduction in epidermal growth factor (EGF) receptor recognition at the cell surface, coupled with an alteration in the binding characteristics of EGF for its specific receptors. Specifically, the heterogeneity of binding exhibited by EGF was affected, and there was loss of the high affinity binding component. EGF-induced autophosphorylation of the EGF receptor was unaffected by IFN treatment. The trafficking of EGF-receptor complexes was followed using three-dimensional confocal microscopy. Confocal imaging revealed that the rapid internalization of EGF-receptor complexes was significantly reduced when cells were exposed to IFN. Accompanying the IFN-induced changes in receptor binding characteristics, we identified an alteration in EGF receptor gene expression; when cells were treated with IFN-alpha, elevated RNA levels specific for the EGF receptor were detected. Overall, IFN-alpha treatment inhibited EGF-induced cell proliferation. Our results imply that EGF-bound receptors that are unable to internalize are not fully competent with respect to signal regulation of both gene expression and growth. The data suggest that the signaling potential of the bound growth factor-receptor complex is apparently increased by an unspecified, species-specific, high affinity binding component. We propose that IFN treatment of responsive cell prevents the interaction of EGF-bound receptor with this component.

Cytoskeletal organization following cannabinoid treatment in undifferentiated and differentiated PC12 cells.

Confocal microscopy in association with three-dimensional reconstruction was used to examine the changes in the microtubules and microfilaments following cannabinoid treatment of PC12 cells. Microtubules and microfilaments were disrupted in a dose-dependent manner following treatment with 10-30 microM delta 9-tetrahydrocannabinol (THC). A disruption of microtubules and microfilaments was observed following treatment with 30 microM cannabidiol and cannabinol. The amount of microtubules and microfilaments was reduced in a dose-dependent manner following treatment with 10 and 20 microM THC. Cannabidiol and cannabinol reduced the amount of microtubules and microfilaments; however, the reduction was less than that observed with THC treatment. Following the addition of nerve growth factor, differentiated PC12 cells were generally more sensitive to cannabinoid treatments than undifferentiated cells. The possible mechanisms that may account for the changes in microtubules and microfilaments following cannabinoid treatment are discussed.

Irregular geometries in normal unmyelinated axons: a 3D serial EM analysis.

Axons have generally been represented as straight cylinders. It is not at all uncommon for anatomists to take single cross-sections of an axonal bundle, and from the axonal diameter compute expected conduction velocities. This assumes that each cross-section represents a slice through a perfect cylinder. We have examined the three-dimensional geometry of 98 central and peripheral unmyelinated axons, using computer-assisted serial electron microscopy. These reconstructions reveal that virtually all unmyelinated axons have highly irregular axial shapes consisting of periodic varicosities. The varicosities were, without exception, filled with membranous organelles frequently including mitochondria, and have obligatory volumes similar to that described in other neurites. The mitochondria make contact with microtubules, while the other membraneous organelles were frequently found free floating in the cytoplasm. We conclude that unmyelinated axons are fundamentally varicose structures created by the presence of organelles, and that an axon's calibre is dynamic in both space and time. These irregular axonal geometries raise serious doubts about standard two dimensional morphometric analysis and suggest that electrical properties may be more heterogeneous than expected from single section data. These results also suggest that the total number of microtubules contained in an axon, rather than its single section diameter, may prove to be a more accurate predictor of properties such as conduction velocity. Finally, these results offer an explanation for a number of pathological changes that have been described in unmyelinated axons.

Reconstructive three-dimensional electron microscopy. A routine biologic tool.

Twenty years ago a laboratory could devote an entire year or more to the collection and analysis of a single set of serial electron micrographs. In contrast, simple technical improvements have now made it possible to take embedded material and have in hand complete computer reconstructions of cells' organelles, microtubules, etc., in less than a week. With a few additional minor improvements, this time could be reduced to only two or three days. Experience in our laboratory suggests that almost without exception these reconstructions provide new insights into both the structure and function of cells. We illustrate this point by presenting a new, unpublished anatomic feature of mammalian nuclei, the "nuclear tube." This example is typical of many other unpublished incidental findings we have made over the last five years using serial electron microscopy as a routine tool, and we believe it represents only the tip of a largely unexplored world of three-dimensional cytoarchitecture.