The developing chick limb was studied to determine the ability of parathyroid hormone (PTH) and prostaglandin E2 (PGE2) to increase intracellular cyclic AMP (cAMP) during various stages of development. All developmental stages examined (stages 20-21, 24-25, and 26-28) responded to PGE2 when the cells were assayed immediately following the removal of the limbs from the embryos. In contrast, only stage 26-28 limb cells responded to PTH when assayed in a similar manner. The response to PTH was temporally correlated with the appearance of cartilage matrix in vivo. Undifferentiated limb cells were also cultured and assayed at various times for hormone responsiveness. Stage 24-25 high-density cell cultures responded initially to PGE2 but not to PTH. However, by 36 h and in all subsequent time intervals tested, the response to PTH was significantly greater than that to PGE2. The PTH receptor, in contrast to that of PGE2, was shown to be sensitive to trypsin treatment, but could be generated during subsequent cell culture. The majority of the hormone-responsive cells were found in cartilaginous regions of the limb, and were shown to respond to both hormones in a dose-dependent manner. The PTH-induced cAMP response was affected by low cell density and mouse serum, both of which significantly inhibit the chondrogenic potential of cultured limb cells. These findings are consistent with a temporal correlation between the development of the PTH response and chondrogenesis in vivo.
While parathyroid hormone (PTH), calcitonin (CT), and certain prostaglandins (PGs) are known to regulate the metabolism of both osteogenic and osteolytic cells of the adult skeleton through an adenosine 3', 5'-monophosphate-dependent mechanism, little is known about the development of this hormonally mediated response in embryonic skeletal tissues. In the present study, the responsiveness of embryonic skeletal elements to PTH and PGE2 was examined during various stages of development utilizing cAMP concentrations as an indicator of hormone-receptor interaction. The cytology of the limb skeletal system was examined also at each stage tested in order to compare the differentiated cellular phenotypes with their hormonal responsiveness. Prior to differentiation of cartilaginous elements in developing limb buds (stage 20-21), cells were responsive to PGE2 and epinephrine (EPI) but not to PTH. The first consistent response to PTH occurred coincident with the initial differentiation of the cartilage phenotype in limb buds (stage 24-25). A responsiveness to both PTH and PGE2 was progressively increased as maturation of cartilaginous and osteogenic elements occurred (stage 26-35). The initial response to CT was detected within cartilage rods in which osteogenic cells had differentiated (stage 33-35). The results of this study indicate that PGE2-sensitive cells exist within the developing limb prior to cytodifferentiation. The development of PTH responsiveness within embryonic chick limb buds is correlated with the onset of both chondrogenesis and osteogenesis in vivo.
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