Biomedical subjects

C V Gay

Publications and source records attributed to C V Gay.

At least 37 records · Page 2Linked to original sources

PubMed1997

Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol.

This study assessed mitochondrial membrane potential changes in cultured osteoblasts treated with hormones known to regulate osteoblasts. A fluorescent carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide, also called JC-1, was used as a probe. JC-1 emits photons at 585 nm (orange-red) when the membrane potential in mitochondria is highly negative, but when the potential becomes reduced emission occurs at 527 nm (green). Osteoblasts were rinsed in serum-free medium for 5 min, then loaded with 1 x 10(-6) M JC-1 for 10 min. The distribution and intensity of JC-1 fluorescence were evaluated with a laser-scanning confocal microscope system. Hormone treatments included parathyroid hormone (PTH; 10(-8) M), 17beta-estradiol (10(-8) M), and thyroxine (T4; 10(-8) M). The potassium ionophore valinomycin (10(-6) M) was used as a control since it is known to disrupt the electrochemical gradient of mitochondria without interfering with the pH gradient. Valinomycin caused a profound, rapid increase (22.5% above untreated values) in the green/red ratio, which indicated a lowering of the mitochondrial membrane potential in all samples evaluated. PTH caused a less pronounced, but significant (7-14%), reduction in membrane potential in all cells examined. PTH is known to affect osteoblasts in a number of ways and is inhibitory to mitochondrial respiration; the results confirm this effect. For estradiol, half of the cells responded at a significant level, with a membrane potential reduction of 6 to 13% being recorded; the other half did not respond. Thyroxine did not alter mitochondrial membrane potential. Responses were detectable within 20 s for valinomycin, but occurred at a slower rate, over 200 to 300 s, following PTH and estradiol treatment. Responses to PTH and estradiol could be due to mitochondrial uptake of cytosolic Ca2+.

Animals↗

PubMed1997

Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts.

The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates Gs-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with Gi- and Go-proteins, blocked both 10(-8) M and 10(-11) M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein alpha subunits, revealed a 48 kDa Gs alpha, a 41 kDa Go alpha, a 34 kDa Gi alpha-3, and a unique 68 kDa G alpha subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a Gs alpha (48 kDa) and a Go alpha (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a Gs and a pertussis toxin-sensitive G-protein (Go and/or Gi alpha-3).

Acids↗

PubMed1997

Chondrocytes of the tibial dyschondroplastic lesion are apoptotic.

Tibialdyschondroplasia (TD) is a disease characterized by the formation of an avascular, non-mineralized lesion along the mature face of the epiphyseal growth plate in rapidly growing chickens. In the normal growth plate, cells progress from a proliferative phase to hypertrophy where the tissue is vascularized and replaced by trabecular bone. In TD, cells apparently cease their development early in the transition to hypertrophy. These diseased cells are not removed by vascularization nor does mineralization occur. The resulting lesion increases in size as proliferative cells continue to divide in the absence of removal and replacement of cartilage by bone. This laboratory has previously reported that cells of the TD lesion have the morphological appearance of necrotic cells or in some cases apoptotic cells. In this study we examine in more detail the status of cells comprising the TD lesion using molecular techniques. Genomic DNA isolated from cells of severe TD lesions show the nucleosomal laddering indicative of apoptosis, while DNA isolated from proliferative and hypertrophic cells does not. This result was confirmed by the use of the Cell Death Detection ELISA which shows quantitatively that cells from severe TD lesions contain nearly twice as many nucleosomal fragments as cells from the hypertrophic zone while proliferative chondrocytes do not have significant fragmentation. In situ examination of the epiphyseal growth plate with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) clearly shows that the cells of the severe TD lesion are apoptotic. Cells from smaller lesions are stained to a lesser extent or not at all by TUNEL. We believe that the apoptosis seen in TD is a secondary effect of the disease and not its primary cause.

Animals↗

PubMed1996

Basic fibroblast growth factor: an autocrine growth factor for epiphyseal growth plate chondrocytes.

Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue.

Animals↗

PubMed1996

Role of microscopy in elucidating the mechanism and regulation of the osteoclast resorptive apparatus.

Microscopic studies have assisted in revealing some of the components of the resorptive apparatus of osteoclasts, specifically carbonic anhydrase and the proton-translocating ATPase. Further, microscopy has helped substantiate the types of proteolytic enzymes secreted into the resorption lacuna. Regulatory agents affecting the resorptive process in vitro include parathyroid hormone, 17 beta-estradiol, calcitonin, and 1,25-dihydroxyvitamin D3. Studies showing the specific binding of parathyroid hormone, estradiol, and calcitonin to osteoclast plasma membrane are discussed. While specific binding suggests that direct effects may occur, further investigation is needed to substantiate this possibility.

Animals↗

PubMed1996

Immunolocalization of osteonectin in avian tibial dyschondroplastic cartilage.

Osteonectin is an acidic calcium-binding protein found in cartilage, bone matrix, vascular endothelium, and areas of tissue repair. Using immunocytochemistry, osteonectin has been localized in all zones of the normal avian epiphyseal growth plate with notably high amounts in the hypertrophic zone. In the proximal portion of this zone the staining was intracellular, while in the distal calcifying portion of the hypertrophic zone staining was both intracellular and extracellular. Osteonectin was also detected in the growth plate associated with lesions of chickens with tibial dyschondroplasia (TD). Intense intracellular staining was observed in hypertrophic chondrocytes proximal to the lesion; staining was markedly diminished in the TD lesion; extracellular matrix was devoid of staining. Staining intensity was high along the peripheral edges of the lesion that were undergoing vascularization and resorption. This was the only area in the dysplastic cartilage where staining was observed in the extracellular matrix as well as intracellularly. Similar patterns were viewed in all TD lesions examined, whether they were spontaneous or induced by dietary treatments or genetic selection.

Animals↗

PubMed1996

Development of a new method for obtaining osteoclasts from endosteal surfaces.

Techniques for the isolation of a highly pure population of viable osteoclasts are limited. For this reason, we developed an isolation procedure that results in a high yield of osteoclast-like cells, up to 92% pure, from 3-wk-old chicken tibias. The unique feature of the method is the migration of cells from marrow-free endosteal surfaces to vitronectin-coated plates. The cells retain the osteoclast phenotype and remain viable in culture for a minimum of 1 wk. The cells were characterized and compared to two populations of authentic avian osteoclasts, which were isolated on the basis of association with fibronectin-coated plates. The cells contained substantial amounts of tartrate-resistant acid phosphatase. Alkaline phosphatase levels were negligible, suggesting little contamination by osteoblasts. Response to parathyroid hormone, dibutyryl cyclic adenosine monophosphate, calcitonin, acetazolamide, 17 beta-estradiol, and prostaglandin E2 was evident, as detected by measuring acid production. The vitronectin-associating cells contained numerous mitochondria, had the ability to resorb bone in an in vitro bone slice assay, and specifically bound biotinylated vitronectin. At 5 d of culture, the cells demonstrated marginal multinuclearity, having two to three nuclei. A large number (approximately 1 x 10(6) cells/tibia) of viable cells that exhibit characteristics of authentic osteoclasts can be obtained by the method described. Potentially, this method could be applied to other species.

Acid Phosphatase↗

PubMed1996

Immunolocalization of basic fibroblastic growth factor in avian tibial dyschondroplastic cartilage.

Recent research in our laboratory has demonstrated that basic fibroblast growth factor (bFGF) is a permissive mitogen for epiphyseal growth plate chondrocytes. Immunocytochemistry demonstrated the presence of bFGF in the proliferative and hypertrophic zones of normal epiphyseal growth plates of 4-wk-old broiler chickens. The purpose of this investigation was to extend this research to include examination of the status of bFGF in the cartilage lesion associated with tibial dyschondroplasia (TD). Immunocytochemistry revealed that the distribution of bFGF in the growth plate proximal to the TD lesion was similar to that observed with normal growth plate. However, the intensity of immunofluorescence was greatly diminished in the TD lesion. The number of chondrocytes staining positive for bFGF was also reduced. In the peripheral edges of the lesion where cartilage was being actively resorbed, the staining intensity was greatly increased when compared to the rest of the TD lesion. Similar patterns were observed in all TD tissues examined whether the lesions were spontaneous or induced by dietary treatments or genetic selection. It is hypothesized that the decrease in bFGF, a potent angiogenic factor, may be responsible for the poor vascularization of the TD lesion.

Animals↗

PubMed1995

Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophilic calcium-sensitive fluorescent dye, calcium green C18.

The synthesis of Calcium Green C18, a lipophilic fluorescent calcium-sensitive dye, and its use as a monitor of Ca2+ efflux from cells is described. This indicator consists of a Calcium Green-1 molecule conjugated to a lipophilic 18-carbon alkyl chain which will intercalate into cell membranes. The Kd of the indicator for Ca2+ in aqueous solution (pH 7.2, 22 degrees C, ionic strength 0.1 M) is 0.23 +/- 0.04 microM and in the presence of liposomes is 0.062 +/- 0.007 microM. Due to its high negativity, the calcium chelating fluorophore faces the cell exterior, when loaded under a defined set of conditions. The dye was found largely on the surface of the cells when loaded at a concentration of 5 microM for 10 min at 37 degrees C. Five minutes after introduction of EGTA, 83-95% fluorescence disappeared, indicating that most of the fluorophore was on the cell surface. Photobleaching was minimal (3-13%). A confocal laser scanning microscope was used to detect and quantify fluorescence. Internalized dye was apparent in cells loaded for longer times (30-60 min) and in membrane-impaired cells, as shown by uptake of propidium iodide. Under defined confocal laser scanning microscope settings, a transient fluorescence at the periphery of approximately 30% of the cells was observed following 10(-8) M parathyroid hormone treatment, indicating the presence of outwardly directed calcium transport across the plasma membrane. Calcium efflux usually lasted 7-10 min, peaking at around 2-3 min. Changes in cell shape were also observed. Calcium efflux was shown to be sensitive to (a) 10 microM quercetin and 10 microM vanadate, partially specific inhibitors of plasma membrane Ca(2+)-ATPase, to (b) 0.1 mM trifluoperazine, an agent which renders calmodulin ineffective, and to (c) 10 mM neomycin sulfate, which blocks release of Ca2+ from intracellular stores. Thapsigargin (5 microM), an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, prolonged fluorescence. These observations indicate that cell surface fluorescence was due to the capture of Ca2+ by Calcium Green C18 after Ca2+ had been translocated across osteoblast plasma membranes. Involvement of the plasma membrane Ca(2+)-ATPase, known to be present in osteoblasts in substantial amounts, is implicated.

Animals↗

PubMed1995

Characterization of calcium efflux by osteoblasts derived from long bone periosteum.

Inside-out vesicles were prepared from the plasma membrane of osteoblasts which had been isolated from the periosteum of 2-3-week-old chicken tibia and cultured for 6-8 days. Calcium uptake was initiated by adding 0.4 mM ATP and detected as a reduction in fluorescence from the reaction medium using the Ca(2+)-specific fluoroprobe, fluo-3. The reaction medium contained ouabain (1 mM) to block Na+, K(+)-ATPase activity and oligomycin (20 micrograms/ml) to block mitochondrial activity. Thapsigargin (5 microM) had little effect, indicating that contributions to Ca2+ uptake by endoplasmic reticulum derived microsomes were minimal. The Ca2+ uptake rate was 9.9 +/- 2.3 nmol/mg protein/min. Trifluoperazine (0.1 mM), which impairs the capacity of calmodulin to activate Ca(2+)-ATPase, substantially inhibited transport, as did quercetin (10 mM) and vanadate (10 microM), inhibitors of Ca(2+)-ATPases. This study has shown the presence of an outwardly directed, calmodulin-sensitive calcium transport system in the primary osteoblast plasma membrane. The pumping rate is substantially less than rates found in the intestine, a tissue which is involved in massive transport of Ca2+, but is similar to rates found in many other tissues. It is concluded that the enzyme does not support calcium translocation to sites of mineralization.

5'-Nucleotidase↗

PubMed1994

Specific binding of estrogen to osteoclast surfaces.

Specific plasma membrane binding sites for 17 beta-estradiol were shown to exist on avian osteoclasts by the use of a fluorescent conjugated estrogen, 17 beta-estradiol-6-carboxymethyloxime- bovine serum albumin-fluorescein isothiocyanate. Binding was dose and time dependent, as well as specific, since 17 beta-estradiol blocked the binding of the complex. Tamoxifen was also an effective blocking agent. Rapid responses to 17 beta-estradiol included decreased acid production (41.5%) and distinct cell shape changes.

Acids↗

PubMed1994

Isolation and localization of basic fibroblast growth factor-immunoreactive substance in the epiphyseal growth plate.

Previous research in our laboratory has shown basic fibroblast growth factor (bFGF) to be a permissive mitogen for isolated avian growth plate chondrocytes. The present study was conducted to determine whether bFGF is present in avian growth plate and, if present, to determine its localization within the tissue. Immunohistochemical studies revealed that bFGF is present in the resting proliferative and hypertrophic calcifying zones of the growth plate but is absent from the prehypertrophic zone. Basic FGF appears to be associated with the extracellular matrix of the proliferative zone, but it is predominantly intracellular in the hypertrophic and mineralizing zone chondrocytes. Partial purification of cartilage-derived bFGF was performed on crude extracts of cartilage using heparin-Sepharose affinity chromatography. The presence of bFGF in the heparin-Sepharose column fractions was confirmed by immunoblotting and radioimmunoassay. Furthermore, western blot analysis of the extracts showed multiple protein bands having bFGF immunoreactivity, in the molecular weight range 14.4-18 kD. The data support the hypothesis that bFGF has a dual role in the growth plate. In the proliferative zone it acts as a chondrocyte mitogen, whereas when released from terminal hypertrophic chondrocytes, bFGF may serve as a chemotactic signal for metaphyseal blood vessel proliferation.

Animals↗

PubMed1994

Characteristics and culture of osteoblasts derived from avian long bone.

A method is presented for isolating primary osteoblasts from the periosteal surface of chick tibia. The culture system identified supports both cell proliferation and phenotype retention. Cell numbers increased 8-fold in Week 1 and 20-fold over a total of 12 days. Well-established osteoblast markers, alkaline phosphatase staining, gamma-carboxyglutamic acid, osteocalcin, type I collagen, and parathyroid hormone binding were detected. Osteocalcin, gamma-carboxyglutamic acid, and type I collagen were present on culture Day 4, and were increased in amount by Day 8, but were similar to the earlier level on Day 12, suggesting that the phenotype may revert to a less differentiated state by 12 days in culture. Alkaline phosphatase staining was intense at all three assay times, however. During the last 4 days of the 12-day culture period, proliferation rates were higher than in the previous 8 days.

Alkaline Phosphatase↗

PubMed1994

Surface binding and clearance of calcitonin by avian osteoclasts.

Osteoclasts, isolated from chick tibias and maintained in culture for 4-6 days, were treated with biotinylated calcitonin followed by fluorescent avidin. In 2 min the entire cell surface fluoresced, by 7.5 min fluorescence was centrally clustered and by 10 min fluorescence had diminished to background levels. Specific binding was blocked by excess unlabeled calcitonin and not influenced by unrelated peptide. The results show that avian osteoclasts bind calcitonin specifically and that the bound calcitonin is rapidly cleared from the cell surface, which indicates involvement of receptor-mediated endocytosis.

Animals↗

PubMed1994

Vitamin K deficiency does not functionally impair skeletal metabolism of laying hens and their progeny.

The purpose of this research was to determine the effect of vitamin K deficiency on indices of skeletal metabolism in laying hens, developing embryos and young growing chickens. Laying hens were fed a vitamin K-deficient diet for 28 wk, which resulted in impaired blood clotting and reduced bone gamma-carboxyglutamic acid (Gla) concentration compared with vitamin K-sufficient hens. However, this treatment did not influence egg production, eggshell deposition or other reproductive performance criteria. Vitamin K-deficient embryos were able to mobilize sufficient quantities of calcium for normal skeletal development, although they exhibited severe reduction in blood clotting and bone Gla concentration. Similar results were obtained from progeny of both vitamin K-sufficient and -deficient hens fed deficient diets for 4 wk after hatching. These results indicate that a severe reduction in skeletal protein Gla concentration does not interfere with normal development of this tissue.

1-Carboxyglutamic Acid↗

PubMed1993

Effect of estrogen on acidification in osteoclasts.

Isolated chicken osteoclasts were neutralized with NH4Cl, then recovery of acid formation was monitored by measuring acridine orange fluorescence over a period of 4 hrs. Estradiol-17 beta and diethylstilbestrol (DES), in nanomolar amounts, blocked acidification 20% to 60%. Effects were detectable in 30 min and maximal by one hr. Estradiol-17 alpha was ineffective. This action of estradiol is believed to be mediated at the plasma membrane since the response was rapid and was not affected by cycloheximide inhibition of protein synthesis. Stimulation of acidification by PTH and cyclic AMP was blocked by estradiol. Estradiol was inhibitory to the same extent as calcitonin; these effects were not additive. Estradiol-17 beta and DES, in micromolar but not in nanomolar amounts, blocked proton pumping in isolated plasma membrane vesicles.

Acridine Orange↗

PubMed1993

Evidence that estrogen binding sites are present in bone cells and mediate medullary bone formation in Japanese quail.

Studies were carried out in Japanese quail to characterize the binding of estrogen to bone cells and to determine whether induction of medullary bone by estrogen is mediated by estrogen receptors. First, attempts were made to identify specific, high affinity nuclear binding sites for estrogen with a nuclear exchange assay in oviduct and femora from laying female quail and in liver, kidney, and femora from adult male quail treated with estradiol valerate (2 mg per bird). High-affinity nuclear estrogen binding sites were detected in each of the tissues except femora. Second, estrogen binding sites in femora from male quail were localized by radioautography after treatment with 100 microCi of [3H]-17 beta-estradiol ([3H]-E). Estrogen binding sites were present at 1 and 3 h after administration of [3H]-E, and binding of the radiolabeled hormone was prevented by the simultaneous administration of an excess of radioinert estrogen. Third, estradiol valerate (.4 mg) was given as a bolus to adult male Japanese quail. Five days later, the quail had hypercalcemia due to accumulation of phosvitin in serum, had an extensive network of medullary bone at the femur midshaft, and had reduced cortical bone area. The nonsteroidal, anti-estrogen tamoxifen (2 mg/day) prevented estrogen-induced hypercalcemia, medullary bone formation, and reduction of cortical bone area. Fourth, the estrogen target cells in femora from adult male quail that were induced to differentiate to osteoblasts by estrogen treatment were located by [3H]-proline radioautography as early as 12 h following administration of the hormone. These results are interpreted as evidence that induction of medullary bone is a process mediated by estrogen receptor.

Animals↗

PubMed1992

Specific binding of parathyroid hormone to living osteoclasts.

We show that osteoclasts bind parathyroid hormone (PTH) in a manner that displays the properties of receptor-dependent hormone binding, that is, saturability, time dependence, temperature dependence, and hormone specificity. Osteoclasts were isolated from the endosteum of 2 to 3 week chick tibiae and maintained in culture for 4-6 days. Bovine PTH-(1-84) was biotinylated with N-hydroxysuccinimidobiotin. Biotinyl-PTH (btPTH, 10(-5)-10(-11) M) was added to the cultured osteoclasts for 2-20 minutes. After rinsing away unbound btPTH, fluorescein isothiocyanate-labeled avidin (FITC-avidin) at a concentration of 66 micrograms/ml was applied. Receptor binding characteristics were assessed: (1) saturation occurred at around 10(-6) M btPTH; (2) competition of excess unlabeled PTH was found, namely, a 10-fold excess abolished fluorescence; (3) specificity was shown by adding other polypeptide hormones (insulin, glucagon, and calcitonin) in 10- to 100-fold excess--no effect on PTH binding was observed; and (4) affinity of btPTH for its binding site was indicated by half-maximal binding approximately equal to 10(-7) M for both osteoclasts and osteoblasts. Biotin (10(-5) M) or FITC-avidin (66 micrograms/ml) alone did not cause fluorescence. The time course of btPTH on the cell exterior was short: at 2 and 5 minutes dots of fluorescence were randomly dispersed over the cell surface, by 10 minutes most of the fluorescence was clustered in one region of the membrane, and by 20 minutes most of the hormone was no longer present on the surface of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Avidin↗