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.
We tested the effects of various parathyroid hormone (PTH) peptides on alkaline phosphatase (ALP) activity in the osteoblastic cell line ROS 17/2.8. In dexamethasone-treated ROS 17/2.8 cells there was a dose-related increase in ALP activity due to treatment with hPTH (53-84). ALP activity was stimulated by 10 nM hPTH (53-84) by a mean of 1.51 +/- 0.07-fold (P less than 0.001) in nine experiments, whereas the same dose of bPTH (1-34) and bPTH (1-84) inhibited enzyme activity to 0.36 +/- 0.02-fold (P less than 0.001) and 0.37 +/- 0.03-fold (P less than 0.001), respectively. Significant stimulation of ALP activity occurred with doses of hPTH (53-84) as low as 0.01 nM. There was no stimulation of enzyme activity by hPTH (53-84) in the absence of dexamethasone; the maximum ALP response to hPTH (53-84) occurred between 96 and 144 hours, and no significant effect was seen at time periods less than 96 hours. The optimum dose of dexamethasone required to enable the response to hPTH (53-84) was 10 nM. Carboxylterminal PTH fragments had a specific stimulatory effect on ALP activity in dexamethasone-treated ROS 17/2.8 cells, but the aminoterminal PTH effect appeared to be dominant, as the equimolar combination of bPTH (1-34) and hPTH (53-84) resulted in inhibition of ALP activity. Thus, in order for the effects of carboxylterminal fragments to be manifest, the cells would have to be stimulated under conditions in which the aminoterminal receptor is unoccupied; this could occur under some in vivo conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
We have previously shown that osteoblasts derived from trabecular bone explants and cultured long term in 10 nM dexamethasone ((HOB + DEX) cells) exhibited properties consistent with a more differentiated phenotype compared with those grown in the absence of dexamethasone ((HOB-DEX) cells). To characterize these two cell models further, we measured the steady-state mRNA levels of the phenotypic markers alkaline phosphatase (ALP), collagen type I (COLL) and osteocalcin (OC), OC production, and the activities of ALP and parathyroid hormone (PTH)-stimulated adenylate cyclase. These findings were then correlated with the age and sex of the bone donors. Long-term culture in dexamethasone significantly increased ALP and OC mRNA levels and the activities of ALP and PTH-stimulated adenylate cyclase but not OC production, in (HOB + DEX) compared with (HOB-DEX) cells (p < 0.05). When the data were examined with respect to the age of the bone donor, age-dependent differences in the expression and responses to dexamethasone were apparent. ALP and PTH-stimulated adenylate cyclase activities decreased with increasing age of the bone donor in (HOB-DEX) and (HOB + DEX) cells (p < 0.05). There were no significant correlations between phenotypic marker mRNA levels and bone donor age in (HOB-DEX) and ((HOB + DEX) cells. All age-dependent decreases in ALP and PTH-stimulated cyclase activities were enhanced in the (HOB + DEX) cells. However, when the data were examined according to the sex of the bone donor, there were no differences in mRNA levels, OC production, or ALP and cyclase activities between cells from male and female donors. These results indicate an age dependence in the expression of osteoblastic markers in human bone cells at different stages of differentiation: thus osteoblastic cultures derived from older donors are likely to contain fewer osteoprogenitor cells, lower levels of glucocorticoid receptors or represent more differentiated osteoblasts compared with those derived from younger donors.
Nitric oxide is a biological mediator that regulates blood vessel wall tonus, enhances macrophage cytotoxicity, and inhibits cellular immune reactivity. Primary acute rejection is associated with increased intragraft production of NO but it is unknown whether this delays or enhances the loss of graft function. The aim of the current study was to determine the effect of L-NAME, a nitric oxide synthase inhibitor, on the course and histopathology of rat cardiac allografts with primary acute rejection. L-NAME decreased the graft survival time from 9.4+/-1.5 to 6.9+/-0.3 days; the histopathology at asystole showed predominantly ischemic necrosis. L-NAME combined with antihypertensive drugs restored the rejection time (from 8.6+/-0.4 to 14.2+/-3.2 days) and resulted in an acute rejection pattern. We conclude that blocking of nitric oxide formation during acute rejection of a vascularized cardiac graft results in a decreased graft survival time and ischemic graft necrosis, very likely secondary to unopposed vasoconstriction.
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