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Biomedical subjects

L G Rao

Publications and source records attributed to L G Rao.

At least 37 records · Page 2Linked to original sources
PubMed1989

Dog and rat kidney proximal tubule cells in culture: responses to parathyroid hormone.

We have described and compared culture systems for proximal tubule cell (PTC) preparations from dog and rat kidney. Cells were prepared from kidney cortex by enzyme digestion and purified on Percoll density gradient. The dog PTC and rat PTC differed in their growth characteristics in culture. Although the dog PTC tended to overgrow the contaminating fibroblastic cells, the rat PTC tended to be overgrown by the latter cells when cultured in medium containing 15% fetal calf serum (FCS). Cultures of rat PTC in serum-free medium or medium containing only 2% FCS yielded only epithelium-like cells exhibiting characteristics of cells that are proximal tubular in origin. These properties include protrusion of microvilli, high alkaline phosphatase activity, ability to transport sugar, and responsiveness to parathyroid hormone (PTH) in terms of cAMP production. The time course and dose-response curves of PTH-stimulated cAMP accumulation were studied in dog and rat PTC. The estimated half-maximal concentrations (Kact) for PTH in dog and rat PTC were 1.2 and 100 nM, respectively. Both values are within the range reported in the literature for the respective renal membrane preparations. In addition to our previously reported data on dog PTC, this study revealed the presence of PTH-inhibitable 25-hydroxyvitamin D3-24-hydroxylase in dog PTC. These two model cell culture systems should prove useful in studying PTH action in renal cells.

24,25-Dihydroxyvitamin D 3↗
PubMed1989

Calcium and protein kinase C enhance parathyroid hormone- and forskolin-stimulated adenylate cyclase in ROS 17/2.8 cells.

Both parathyroid hormone (PTH)- and forskolin-stimulated adenylate cyclase activities in ROS 17/2.8 cells are enhanced by increasing the medium concentrations of CaCl2 from 10(-5) M to 3 x 10(-3) M. The ED50 for CaCl2 for both PTH- and forskolin-stimulated activities are similar. The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, also enhanced both PTH- and forskolin-stimulated adenylate cyclase. This action of PMA is specific for protein kinase C as phorbol esters that are not activators of protein kinase C had no effect on the system. The combined effects of PMA and CaCl2 were more than additive. The separate and combined effects of PMA and CaCl2 changed the rate of activation of the enzyme (Vmax) but did not modify the ED50 for PTH or for forskolin. PMA and CaCl2 both enhanced the potentiating effect of submaximal dose of forskolin on PTH-stimulated adenylate cyclase. It is concluded that calcium and PMA enhance PTH-sensitive adenylate cyclase and increase the production of cAMP by a mechanism that appears to involve the catalytic subunit of the enzyme and probably its interaction with a guanine nucleotide regulatory protein.

Adenylyl Cyclases↗
PubMed1989

Calcium modulation of the parathyroid hormone-sensitive adenylate cyclase in ROS 17/2.8 cells: effects of N-(6-aminohexyl-5-Cl-naphthalene sulfonamide) (W-7) and trifluoperazine (TFP).

The calcium modulation of the cyclic 3',5'-adenosine monophosphate (cAMP) response to parathyroid hormone (PTH) was studied in a clonal osteosarcoma cell line ROS 17/2.8. CaCl2 was found to stimulate the PTH-sensitive cAMP response of intact cells. At the maximal concentration of 1 mM CaCl2, the maximum response to PTH was increased, but the ED50 for PTH and the time course of maximal cAMP production were not affected. Verapamil blunted, while the cation ionophore A23187 enhanced, the stimulatory effect of CaCl2. Trifluoperazine (TFP) and N-(6-aminohexyl-5-Cl-naphthalene sulfonamide) (W-7) inhibited the stimulatory effect of CaCl2. In membranes prepared in the presence of 0.1 mM CaCl2, a biphasic effect of CaCl2 was demonstrated: stimulation at concentrations of 60-100 microM, and an inhibition above 200 microM, when adenylate cyclase was assayed in the presence of 200 microM EGTA. Addition of exogenous calmodulin to membranes prepared in the presence of EGTA did not have any effect on the PTH-sensitive adenylate cyclase activity, suggesting that endogenous calmodulin was not effectively stripped from the membranes by EGTA treatment. It is concluded that Ca2+ has both a stimulatory and an inhibitory role in modulating PTH-sensitive adenylate cyclase in ROS 17/2.8 cells by as yet unknown mechanisms, and that the involvement of endogenous calmodulin is implicated.

Adenylyl Cyclases↗
PubMed1989

Human parathyroid hormone carboxyterminal peptide (53-84) stimulates alkaline phosphatase activity in dexamethasone-treated rat osteosarcoma cells in vitro.

Previous studies in our laboratory have demonstrated relatively large numbers of cell surface binding sites for the carboxylterminal (53-84) region of PTH on ROS 17/2.8 rat osteosarcoma cells, a clonal osteoblast-like cell line. In order to gain insight into the significance of these carboxylterminal binding sites, we studied the effect of intact bovine PTH (1-84), its aminoterminal fragment bovine PTH (1-34), and the human PTH carboxylterminal fragment (53-84) on alkaline phosphatase activity in dexamethasone-treated rat osteosarcoma (ROS) 17/2.8 cells. While bovine PTH (1-84) and its aminoterminal 1-34 fragment inhibited alkaline phosphatase activity, we saw a dose-related stimulation of activity by human PTH (53-84), with maximal stimulation occurring after 120 hours, at a concentration of 10(-8) M. The effect was not seen in dexamethasone-untreated cells. To our knowledge, this is the first published demonstration of biological activity of this carboxylterminal PTH peptide, previously thought to be inactive. It is likely that dexamethasone caused differentiation of cells to a type more sensitive to human PTH (53-84). Further studies are necessary to elucidate the physiological significance of these findings.

Alkaline Phosphatase↗
PubMed1988

Effect of long-term and short-term diabetes on the parathyroid hormone sensitive rat renal adenylate cyclase: correlation with vitamin D metabolism.

In short-term experiments, male Wistar rats were made diabetic for 10 days with a single injection of streptozotocin (65 mg/kg body weight). One group of diabetic rats was treated with insulin for 3 days prior to sacrifice. In long-term experiments, vitamin D replete or vitamin D depleted rats were made diabetic for 6 weeks. Criteria for diabetes were loss of weight, glycosuria (Tes-Tape), and hyperglycemia. In long-term diabetic rats the activity of renal mitochondrial 25-hydroxyvitamin D3 (25-(OH)D3) 1 alpha-hydroxylase was significantly decreased and that of 25-(OH)D3 24-hydroxylase increased. However, the parathyroid hormone (PTH) sensitive renal adenylate cyclase activity of diabetic rats was not different from that of the nondiabetic rats in either the vitamin D replete group or the vitamin D depleted group. On the other hand, the PTH-sensitive renal adenylate cyclase activity was significantly higher in short-term diabetic rats than in control and insulin-treated rats. These differences were observed at doses of 10(-8) to 10(-5) M of PTH. This study has demonstrated for the first time that there are differences in the PTH-sensitive adenylate cyclase response between long-term and short-term diabetic rats. The hypersensitivity to PTH of the renal adenylate cyclase observed in short-term diabetic rats probably represents a response to insulin deficiency during the early development of diabetes mellitus in the rats.

Adenylyl Cyclases↗
PubMed1985

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state.

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.

Animals↗
PubMed1985

Binding of intact parathyroid hormone to rat osteosarcoma cells: major contribution of binding sites for the carboxyl-terminal region of the hormone.

Most studies of PTH receptor binding have been carried out with amino-terminal radioligands which only detect binding within that region of the hormone molecule. We studied the binding of electrolytically labeled intact bovine PTH [bPTH-(1-84)], and its amino-terminal fragment, bPTH-(1-34) to intact cloned rat osteosarcoma cells (ROS 17/2.8). We also measured the effects of these hormones on cell cAMP accumulation. Binding equilibrium for the two radioligands was reached by 2 h of incubation at 22 C. However, the cells had higher binding capacity (8-9% or 0.22-0.25 fmol/2 X 10(6) cells) for [125I]bPTH-(1-84) than for [125I]bPTH-(1-34) (4% or 0.11 fmol/2 X 10(6) cells). On the other hand [125I]bPTH-(1-34) bound to ROS cells with higher affinity [dissociation constant (Kd) = 19 nM] than did [125I]bPTH-(1-84) (Kd = 210 nM). Measurements of trichloroacetic acid precipitability and analysis of rebinding of previously incubated radioligand to fresh cells ruled out degradation of the tracer as an explanation for these differences. The maximum cell cAMP response to bPTH-(1-34) (Vmax = 780 +/- 32 pmol/2 X 10(6) cells X 5 min) was reached at 10(-7) M concentration with an affinity [Michaelis-Menten constant (Km)] of 3 nM. On the other hand, the Vmax with intact bPTH-(1-84) was lower (400 +/- 7 pmol/2 X 10(6) cells X 5 min), with a Km of 60 nM). Further studies with the bPTH-(1-84) tracer showed inability of hormonal fragments to compete completely for binding. At a concentration of 3 microM, bPTH-(1-84) reduced tracer binding by 82.5%, compared to 18% by bPTH-(1-34) and 10% by (Nle8,Nle18,Tyr34)bPTH-(1-34)amide, 60% by human PTH (hPTH)-(53-84), and 70% by the combination of bPTH-(1-34) and hPTH-(53-84). hPTH-(53-84) itself did not elicit a cAMP response after 5 min or 1 h of incubation nor did it significantly alter the cAMP response of the cells to bPTH-(1-84). These studies suggest that PTH binds to ROS 17/2.8 cells by sites carboxy-terminal (C-terminal) to position 34, in addition to sites within the amino-terminal portion of the hormone molecule; 72% of the binding of intact hormone to these cells was to the C-terminal 35-84 region of the PTH molecule. The significance of the C-terminal binding sites is presently unclear, but they do not appear to be coupled to adenylate cyclase. Further work is needed to determine the effects of C-terminal PTH fragments on bone cell metabolism.

Amino Acid Sequence↗
PubMed1983

Elimination of cross-reactivity to some component of erythrocytes in an antiserum to parathyroid hormone by absorption with fixed erythrocytes.

Cross-reactivity to some component of rat erythrocytes with an antiserum to parathyroid hormone (anti-PTH) was detected in fixed demineralized sections of bone using the peroxidase-antiperoxidase method. The cross-reactivity was eliminated by the preabsorption of anti-PTH with fixed, washed rat erythrocytes. This technique provides an easy and rapid method with which to eliminate cross-reactivity to erythrocytes whenever such a situation is encountered in immunohistochemical procedures.

Absorption↗
PubMed1983

Immunohistochemical demonstration of parathyroid hormone binding to specific cell types in fixed rat bone tissue.

Deparaffinized sections of fixed decalcified neonatal rat radii were incubated in bovine PTH (bPTH; 1-10 MRC units/ml) or in PTH-solvent. They were then stained for PTH by the peroxidase-antiperoxidase method using guinea pig antiserum to bPTH and the substrate 3,3'-diaminobenzidine-H2O2. Staining caused by nonspecific binding of PTH to the bone matrix and the glass slides supporting the sections was eliminated completely by preincubation of the sections in 100% normal goat serum. Cross-reactivity of the antiserum to erythrocytes was eliminated by preabsorption of the antiserum with fixed rat erythrocytes. After the cross-reactivity of the anti-PTH antiserum to erythrocyte components and the nonspecific binding of PTH to bone matrix were eliminated, we were able to demonstrate intense staining over the cytoplasm of the osteoclasts in rat radii sections incubated with PTH. Less intense staining was observed over the osteocytes, periosteal osteoblasts, and, possibly, the endosteal osteoblasts. An explanation for this differential staining could be that osteoclasts have a greater receptor number and/or a greater receptor affinity for the bPTH than do osteocytes and osteoblasts. This study demonstrates that binding of PTH to bone tissue can be localized in all identifiable osteoclasts, osteocytes, and osteoblasts, and thus suggests that all three cell types can interact directly with PTH.

Animals↗
PubMed1981

Antenatal diagnosis of placental sulphatase deficiency.

A dynamic test for the antenatal diagnosis of placenta sulphatase deficiency was examined in ten patients, selected on the basis of low urinary oestrogen levels (i.e. below the 10th centile at between 30 and 40 weeks gestation). The test consisted of measurement of plasma oestradiol levels over a period of 120 min after an i.v. injection of dehydroepiandrosterone sulphate. The oestradiol response was vigorous in seven patients, absent in two and small, but significant, in one. The three patients with reduced oestradiol response were also found to have raised levels of urinary steroid monosulphates and thus were probably placental sulphatase deficient. The overall clinical importance of placental sulphatase deficiency and the value of the dehydroepiandrosterone sulphate loading test in antenatal diagnosis are discussed.

Adult↗
PubMed1980

Calcium metabolism in a fatal case of sodium fluoride poisoning.

A patient was admitted to a district general hospital within an hour of ingesting a fatal dose of sodium fluoride. The results of laboratory investigations, together with some in vitro findings, support the hypothesis that the hypocalcaemia of fluoride poisoning is the result of fluorapatite formation and not calcium fluoride precipitation, and that its persistence reflects the severity of the calcium deficit and not an inhibitoin of normal homeostatic mechanisms. It is suggested that the role of renal clearance of fluoride may be more important than had been realised hitherto.

Apatites↗
PubMed1980

Specific immunohistochemical localization of type III collagen in porcine periodontal tissues using the peroxidase-antiperoxidase method.

Specific antibodies to porcine gingival type III collagen were raised in sheep. After purification on collagen affinity columns the antibodies were used for immunohistochemical localization of type III collagen in porcine periodontal and dental tissues employing the peroxidase-antiperoxidase (PAP) procedure. The extent of staining of the periodontal tissues was found to approximate the amount of type III collagen measured biochemically. A fairly uniform distribution of type III collagen was observed in the periodontal ligament and gingiva with more intense staining often being associated with blood vessels. A regular pattern of weakly staining fibers could be demonstrated throughout the cementum and in parts of the alveolar bone tissue. In addition, occasional sites in the cementum having a different morphological appearance from the rest of the cementum exhibited bundles of positively stained fibers. Although the bone tissue was essentially unstained, fibers in the endosteal spaces stained strongly. Sharpey's fibers passing from the soft connective tissues into alveolar bone and cementum also stained strongly. Three distinct arrangements of collagen fibers stained by the type III collagen antibodies could be identified: first, a reticular pattern, which was seen at the junction of the gingival epithelium and connective tissue, and in the endosteal spaces and dental pulp; second, a more diffuse pattern of fibers intermingled with type I collagen in the soft connective tissues; and third, a coating of some Sharpey's fibers, having a core believed to be type I collagen, and of fibers in the cementum inclusions.

Amino Acids↗
PubMed1979

Specific immunohistochemical localization of type I collagen in porcine periodontal tissues using the peroxidase-labelled antibody technique.

Antibody against Type I collagen was raised in rabbits and purified by immunoadsorption on Sepharose-conjugated Types I and III collagen. The cross-reactivity of purified antibody to Type III collagen was found to be less than 0.5% by passive haemagglutination and less than 1.5% by radioimmunoassay. When paraffin sections of fixed and decalcified pig molars were incubated with purified antibody to Type I collagen, varying degrees of staining were observed in the ligament, gingiva, bone and cementum. The periodontal ligament adjacent to bone was more widely stained than that adjacent to cementum in some regions, whereas in others, no difference in staining could be discerned between the two halves of the ligament. The lamina propria of gingiva was stained, and this appeared to be most intense in the vicinity of the overlying epithelium. The fibrous component in the endosteal spaces, the dentine and the extracellular coronal elements in the pulp were generally stained. The impression obtained from the staining pattern is that Type I collagen is not restricted to particular regions of the periodontal ligament or the lamina propria of the gingiva.

Animals↗