Biomedical subjects

C V Gay

Publications and source records attributed to C V Gay.

At least 55 records · Page 3Linked to original sources

PubMed1992

Demonstration of calmodulin-sensitive calcium translocation by isolated osteoclast plasma membrane vesicles.

Plasma membrane vesicles were prepared from chicken osteoclasts, and active calcium transport was demonstrated in a spectrofluorimetric assay using the fluorescent calcium concentration indicator, fura-2. Transport activity was inhibited by quercetin (10 microM), sodium vanadate (10 microM), and the anticalmodulin agents, compound 48/80 (20 and 200 micrograms/ml) and calmidazolium (10 and 20 microM). The transport rate (Vmax, 1.3 nmol/mg protein/min) was not altered in the presence of the protonophore, nigericin (1 microM), indicating that proton transport was not driving calcium transport. Release of accumulated calcium in the vesicles occurred with the addition of bromo-A23187 (5 microM) or ionomycin (5 microM). Increasing calcium transport occurred with increasing calcium concentration. Finally, the calmodulin content of the vesicles was demonstrated to be 54-134 U/mg protein. These results demonstrate that a calmodulin-sensitive, ATP-dependent calcium transporter is present in the osteoclast plasma membrane.

Animals↗

PubMed1991

Ultrastructural localization of tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone.

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Acid Phosphatase↗

PubMed1991

Characterization of the functional stages of osteoclasts by enzyme histochemistry and electron microscopy.

To investigate the functional stages of osteoclasts, the ultrastructural histochemical distribution of the lysosomal enzymes [acid phosphatase (tartrate-sensitive) and neutral phosphatase], the plasma membrane enzymes [alkaline phosphatase, Ca(++)-ATPase, and alkaline ouabain-insensitive p-nitrophenylphosphatase (alkaline p-NPPase)], and the mitochondrial enzyme (cytochrome C oxidase) was evaluated in the chicken tibial metaphysis. Both active-appearing and detached (resting) osteoclasts were studied. Serial sectioning was used to identify detached osteoclasts which were present in the perivascular space. The ultrastructure of detached osteoclasts was similar to that of active osteoclasts, except for the lack of a ruffled border and clear zone, and an altered distribution pattern of small vesicles. Small vesicles were uniformly distributed in the cytoplasm of resting osteoclasts, whereas they were concentrated beneath the ruffled border of active osteoclasts. Alkaline p-NPPase, a marker enzyme for the basal ruffled border, was also apparent on the membrane of small vesicles. However, the vesicles did not possess Ca(++)-ATPase, a marker enzyme for the apical plasma membrane. These findings support the concept that small vesicles serve as a membrane reservoir for the ruffled border membrane. Pre-osteoclasts contained abundant mitochondria and lysosomes, prominent Golgi complexes, moderately developed endoplasmic reticulum, and lacked small vesicles. Pre-osteoclasts appear to fuse with osteoclasts which are attached to the bone surface, but not with detached osteoclasts. The small vesicles, from which the ruffled border arises, are absent from pre-osteoclasts, suggesting that they develop after fusion with pre-existing osteoclasts or after attachment to the bone surface. Alkaline p-NPPase appears to be a marker for differentiation of pre-osteoclasts to mature osteoclasts.

Animals↗

PubMed1991

In vitro resorptive activity of isolated chick osteoclasts: effects of carbonic anhydrase inhibition.

A potent inhibitor of carbonic anhydrase, 5-[3-hydroxybenzoyl]thiophene-2-sulfonamide (HTS), was shown to cause a 37% reduction in the area of resorption pits formed by isolated chick osteoclasts when used at a dose of 10(-7) M. HTS at doses of 10(-9) and 10(-7) M was also effective in reducing acid formation by the osteoclasts (14 and 36%, respectively). Additionally, the effect of HTS was found to be readily reversed by removing the agent, showing that it does not exert a toxic effect on the cells. This study indicates that the inhibitory effect of HTS on bone resorption is at the level of the acid-forming mechanism in osteoclasts and supports the view that carbonic anhydrase has a central role in the process.

Acid Phosphatase↗

PubMed1991

Avian osteoclasts.

Explore the source record for details and available documents.

Acid Phosphatase↗

PubMed1991

Ultrastructural localization of guanylate cyclase in bone cells.

Guanylyl imidodiphosphate (GMP-PNP) hydrolyzing enzyme activity as a means of detecting plasma membrane guanylate cyclase was demonstrated in osteoblasts of chicken tibial metaphysis using a lead citrate histochemical method at the electron microscopic level. Activity was not discerned in osteoclasts or osteocytes. The reaction product development was completely abolished when the sections were incubated with substrate-free or MnCl2-free medium. Guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP) was a less effective substrate than GMP-PNP, and Mn++ was a stronger stimulator than Mg++. No reaction product was observed on the plasma membrane of osteoblasts when beta-glycerophosphate or p-nitrophenylphosphate was used as substrate instead of GMP-PNP. The results implicate guanylate cyclase as a significant effector of osteoblast regulation at the site of the plasma membrane.

Animals↗

PubMed1991

Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts.

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.

Adenosine Triphosphate↗

PubMed1990

Characterization of a Ca2(+)-ATPase in osteoclast plasma membrane.

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+,K(+)-ATPase and 5'-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+,K(+)-ATPase and 5'-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2(+)-ATPase had a high-affinity (KCa = 0.22 microM; Vmax = 0.16 mumol/mg per min) and a low-affinity (KCa = 148 microM; Vmax = 0.37 mumol/mg per min) component. Calmodulin (0.12 microM) had no effect on Ca2(+)-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2(+)-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2(+)-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 microM; Vmax = 0.022 mumol/mg per min) and a low-affinity (Km = 43.8 microM; Vmax = 0.278 mumol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2(+)-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.

Animals↗

PubMed1990

Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles.

A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2(+)-dependent ATP hydrolyzing activity of 0.213 mumol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a KMg of 1.293 microM and Vmax of 0.063 mumol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 microM and a Vmax of 0.232 mumol Pi per mg protein per minute. The Mg2(+)-ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4'-diisothiocyano-2,2'-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3 VO4) at 1 microM had no effect but caused 29.5% inhibition at 1 mM. Na+ could substitute for K+ without loss of activity, NO3- caused 19.5% inhibition when substituted for Cl-, and acetate replacement of Cl- resulted in 36.4% stimulation of Mg2(+)-ATPase. ATP, GTP, ITP, CTP, and ADP were all hydrolyzed effectively. DCCD (0.2 mM), NEM (0.5 mM), nolinium bromide (1 mM), and DIDS (50 microM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3 VO4 (1 mM) had no effect, and duramycin (80 micrograms/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of Cl- resulted in a 46.1 and 55.7% decrease in transport, respectively. ATP supports transport far more effectively than the other nucleotides tested. ADP was ineffective. Experiments using the potassium ionophore, valinomycin, indicated that the proton pump functions electrogenically, with Cl- most likely cotransported by an anion transporter. The proton pump also seems to have at least one anion-sensitive site, elucidated by experiments in the presence of NO3- and Cl-.

Animals↗

PubMed1990

Effects of parathyroid hormone, calcitonin, and dibutyryl-cyclic AMP on osteoclast area in cultured chick tibia.

Slices of osteoclast-enriched endosteal surfaces of 3 week chick tibia were cultured for 1-3 days. Osteoclasts on the bone surface were made visible by acridine orange fluorescence. Osteoclast area was measured by image analysis. Parathyroid hormone (PTH) caused osteoclasts to increase in area about 40%, and calcitonin (CT) caused a decrease in area also of about 40%. Subsequent addition of dibutyryl cyclic AMP to PTH- or to CT-treated cells resulted in a further change of 40 and 30%, respectively. Application of the cyclic AMP analog alone had no effect. All responses were rapid, occurring in 2-4 minutes.

Acridine Orange↗

PubMed1990

Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production.

Effects of interstitial fluid flow on osteoblasts were investigated. Intracellular cyclic adenosine monophosphate (cAMP) levels were monitored in cultured osteoblasts subjected to shear rates ranging from 10 to 3,500 sec-1. Cyclic AMP levels were significantly increased at all shear rates from 1 pmole/mg protein to 10-16 pmole/mg protein. Osteoblasts subjected to a shear rate of 430 sec-1 for 0.5-15 minutes exhibited elevated levels (12-fold) of intracellular cAMP, which were sustained throughout the perfusion period. Osteoblasts were three times more sensitive to flow stimulation than human umbilical vein endothelial cells and baby hamster kidney fibroblasts, which also displayed higher cAMP levels (4-fold) after exposure to flow. To distinguish streaming potential effects from shear stress effects, viscosity was increased 5-fold by addition of neutral dextran to the perfusing medium. Shear stress is a function of viscosity, and streaming potentials are not for a given shear rate. The mechanism of this cellular response to flow was shown to be shear stress dependent. Inhibition of cyclooxygenase by 20 microM ibuprofen completely inhibited the flow-dependent cAMP response, indicating the cAMP response is mediated by prostaglandins. Our results suggest that fluid flow induced by mechanical stress may be an important mediator of bone remodeling.

Animals↗

PubMed1989

Characterization of the cytoskeleton of isolated chick osteoclasts: effect of calcitonin.

We investigated the effects of calcitonin (CT) and parathyroid hormone (PTH) on the distribution of actin, tubulin, vimentin, and on cell size in cultured chick osteoclasts. In addition, we studied the effects of colchicine on intracellular acidity. Osteoclasts were isolated from the endosteum of 2-3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with CT for 30 min or PTH for 60 min and were observed after immunocytochemical staining of cytoskeletal proteins. In untreated cells, actin was found in both a filamentous and a punctate staining pattern, with indented or invaginated regions free of punctate spots. The tubulin distribution in untreated cells was characterized by a pattern of microtubules radiating from the cell center and running parallel to the cell edge. Vimentin staining was usually localized to the perinuclear area. There were no changes in cytoskeletal element distribution or morphology attributable to PTH treatment. Osteoclasts treated with CT were more irregularly shaped, contained more retraction fibers, and were more rounded, with a denser array of cytoskeletal elements in the cell center. In addition, the mean area of the CT-treated cells was significantly less than that of the untreated cells. The actin distribution after CT treatment was still characterized by both a filamentous and a punctate pattern. After CT treatment, vimentin staining appeared more centrally localized than in untreated cells and tubulin staining revealed microtubules which now extended to the retracted cell margin. These results indicate that isolated osteoclasts respond to CT by significant morphological changes which are reflected in the distribution of the major cytoskeletal elements. Disruption of the microtubular system by colchicine treatment also resulted in an initial increase in intracellular acidity, suggesting the involvement of microtubules in the movement of acid-laden vesicles to the exterior.

Acid Phosphatase↗

PubMed1989

Ultrastructural localization of adenylate cyclase in chicken bone cells.

We investigated adenylate cyclase distribution in 6-day-and 3-week-old calvariae and in 6-day-old long bone metaphyses from chickens. Reaction product distribution was on the plasma membrane of osteoblasts, pre-osteoblasts, and forming osteocytes which contacted one another. Osteoclasts and mature osteocytes lacked reaction product. Six-day calvariae reacted less intensely than the other two tissues. In controls, reaction product was markedly diminished or eliminated by removal of forskolin or substrates, or by addition of the inhibitor 2',5'-dideoxyadenosine. The results indicate the importance of cyclic AMP in osteoblast regulation. Osteocytes and osteoclasts may involve alternate mechanisms as major regulatory systems.

Adenylyl Cyclases↗

PubMed1989

Type X collagen in avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is an abnormality of the growth plate of growing chicks characterized by the presence of a mass of nonvascularized, nonmineralized cartilage. To determine if the morphologic changes observed in TD lesions are accompanied by biochemical abnormalities of the cartilage matrix, we performed quantitative and qualitative analyses of collagen in affected cartilage, placing special emphasis on the study of type X collagen, a recently described molecular species that may play a role in endochondral ossification. Collagen from TD lesions of 25-day-old chicks and from growth plate cartilage of normal age-matched chickens and from hypertrophic cones of 1-day-old chicks was sequentially extracted, purified and characterized. No differences were noted in total collagen content and extractibility between these tissues. Determination of the relative amounts of the various collagens extracted showed that type X collagen in the TD lesion was markedly reduced since it was less than half of that found in pure hypertrophic cartilage. These results are consistent with previous morphologic and biochemical studies which suggest a failure of the chondrocytes to fully achieve maturation and hypertrophy. No differences were demonstrated in the CNBr peptide patterns or amino acid composition of type X collagen from normal or TD and hypertrophic cartilages, suggesting that the low content of type X collagen in TD lesion was due to alterations in type X collagen metabolism rather than to structural abnormalities in these molecules.

Amino Acids↗

PubMed1988

Ultracytochemical investigation of calcium-activated adenosine triphosphatase (Ca++-ATPase) in chick tibia.

The ultrastructural distribution of Ca++-ATPase in bone cells of growing chick tibia was investigated by a cytochemical method in order to gain insight into possible sites of calcium ion translocation. Both osteoclasts and osteoblasts showed a polar distribution of reaction product along the plasma membrane. In osteoclasts, enzymatic activity occurred along the portion of the plasma membrane facing the marrow but not along the ruffled border or clear zone. The reaction product in these cells was due solely to Ca++-ATPase action. In osteoblasts, the plasma membrane facing away from bone (apical and lateral membrane) was very intensely stained, whereas the basal membrane was unstained. The reaction product in these cells appeared to be the result of both Ca++-ATPase and Ca++,Mg++-ATPase. In osteocytes, no plasma membrane staining was detectable. Mitochondrial staining in all three types of cells was more sensitive to fixation than was the plasma membrane enzyme, suggesting that mitochondrial and plasma membrane Ca++-ATPases are chemically distinct, as biochemical studies have shown. In general, mitochondria in osteoclasts stained more intensely than those in osteoblasts or osteocytes. Mitochondrial and vesicular sites of activity may be related to intracellular calcium storage, whereas calcium ATPases of the plasma membrane are presumed to be involved in calcium efflux from the cells. Calcitonin treatment did not alter the enzymatic distribution or intensity in osteoclasts. The striking polar distribution of both osteoclast and osteoblast plasma-membrane activity suggests that directional calcium pumping by these cells may be of importance in bone-forming and bone-resorbing mechanisms.

Aging↗

PubMed1988

Characterization of isolated and cultured chick osteoclasts: the effects of acetazolamide, calcitonin, and parathyroid hormone on acid production.

The effects of acetazolamide, calcitonin (CT), and parathyroid hormone (PTH) on acid production in isolated osteoclasts has been investigated. Osteoclasts were isolated from the endosteum of 3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with acetazolamide (10 x 4 M and 10(-7) M), CT (1 mU/ml and 0.31 mU/ml) and PTH (6.5 U/ml and 0.40 U/ml) for 1, 3, 6, and 18 hr. The cells were stained with acridine orange and the intensity of fluorescence measured by a light microscope photometer. Acetazolamide treatment resulted in a steady decline in intracellular acidity, suggesting that carbonic anhydrase plays a major role in acid production in isolated osteoclasts. Treatment with PTH produced a decline in acidity at 1 hr, followed by a peak at 3 hr and then a decline at 6 and 18 hr. The transient increase in acidity may be due to activation of carbonic anhydrase by PTH. Calcitonin treatment also resulted in a decline in cell acidity which was similar, but less pronounced than that resulting from acetazolamide treatment. These results indicate that calcitonin may mediate osteoclast activity by alterations in intracellular acid production.

Acetazolamide↗

PubMed1988

Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution.

The ultrastructure of extracellular membrane-bound matrix vesicles (MVs), their biogenesis, and the surrounding matrix in chick tibial growth plate were studied after quick freezing and freeze substitution (FS) in an organic solvent. There were several notable differences in the ultrastructural preservation of cartilage when FS was used as compared with conventional fixation. The ultrastructural appearance of MVs after FS was extremely variable. Within the MVs, intravesicular filaments, amorphous material, and membrane-associated undercoat structures were observed. Intravesicular filaments, similar in diameter to microfilaments seen in the cytoplasm, were attached to the inside of MV membranes. This observation indicates the similarity of MV membranes and the plasma membrane. In some MVs in the proliferative zone an electron-dense material was present along the inner side of the MV membrane. In the prehypertrophic zone, crystalline material often appeared within the electron-dense material, which may be a precursor form of hydroxyapatite. The earliest crystals observed were in MVs but not in the extracellular matrix. Regarding MV formation, in addition to budding from cell surfaces and to cellular disintegration, this study also indicates that a sequential process of extrusion of preformed cytoplasmic structures may occur. Also, small MVs measuring 25-40 nm seem to arise from the disruption of large MVs. This is a previously unreported observation on MV biogenesis. FS preserves proteoglycans in the cartilage matrix as a fine, filamentous network. Initial extracellular calcification was not associated with this network.

Animals↗

PubMed1988

Ultrastructural analysis of cytochrome oxidase in chick epiphyseal growth plate cartilage.

Histochemical detection of cytochrome oxidase activity in chicken growth plate revealed both positively and negatively stained mitochondria in chondrocytes of all zones, i.e., proliferative, pre-hypertrophic, hypertrophic, and calcifying zones. The proportion of positive to negative cells was lowest in the proliferative zone. As cytodifferentiation progressed, more positively stained cells were present. In positive cells all mitochondria were usually stained, and in negative cells all mitochondria were unstained. A few cells appeared to be in transition and contained both types of mitochondria. The results indicate that chondrocytes utilizing both aerobic and anaerobic metabolism are present in growth plate cartilage and that oxidative metabolism is favored in the more mature cells. The relationship of oxidative metabolism to calcification is discussed.

Animals↗