David M. Biddulph

Chondrogenesis of chick limb mesenchyme in vitro: Effects of prostaglandins on cyclic AMP

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) levels of chick limb bud cells during various stages of chondrogenesis were studied utilizing high density, micro-mass, cell culture. Concentrations of PGE2 in cell cultures at these same stages were measured by radio-immunoassay. Both basal levels of cAMP, as well as PGE2-stimulated changes in cAMP, increased maximally during the first 3 days of culture; this increase was associated with the formation of cell aggregates. Concentrations of PGE2 were also highest during this period. By 6 days of culture, nodules, containing cartilage matrix components, predominated. Both basal levels of cAMP and PGE2-stimulated cAMP levels were significantly decreased at this stage, relative to cultures at day 3. Concentrations of PGE2 fell dramatically in the 6-day cultures containing differentiated cartilage. These results support a regulatory role for both PGE2 and cAMP in the early events associated with chondrogenesis.

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E2 (PGE2) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05-1.0 micrograms/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 micrograms/ml totally inhibiting cartilage formation. Concentrations of PGE2 and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 micrograms/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE2 levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE2 and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.

Development of the cyclic AMP response to parathyroid hormone and prostaglandin E2 in the embryonic chick limb.

SummaryThe 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 itme 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 regenerated during subsequent cell culture. The majority of the hormoneresponsive 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.

Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs

Effects of prostaglandins (PGs) on accumulation of cyclic AMP (cAMP) in the presence of a phosphodiesterase inhibitor were investigated in cells isolated from avian limb buds at various stages of development. Cells were responsive to PGE2 at the earliest stage investigated (stage 20-21) which was well in advance of specific cytodifferentiation of limb tissues. At three later stages (24-25; 26-28; 30-32), the responsiveness of cells isolated from the developing skeletal anlagen of the limb progressively increased coincident with the differentiation and maturation of the cartilage phenotype. Cells isolated from stage 26-28 cartilage rods were responsive also to prostacyclin (PGI2); however, the response produced was only about 50% of the response to an equivalent concentration of PGE2. Cells were not responsive to either PGF2 alpha or 6-keto PGF1 alpha, at concentrations of 30-33 micrograms/ml demonstrating a degree of specificity for PGE2 and PGI2. In the absence of the phosphodiesterase inhibitor, PGE2 increased cAMP accumulation two-fold over the controls and produced a concentration-dependent response between 0.3-30 micrograms/ml. The results demonstrate that PGs are capable of modulating cAMP levels of undifferentiated limb mesenchymal cells as well as embryonic cartilage cells and suggest a role for these compounds in limb chondrogenesis.

Metabolism of cyclic AMP in isolated renal tubules: Effects of prostaglandins and parathyroid hormone

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) with maximal effects of PGE1 being 6-8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2-3 fold greater in tubules exposed to PTH than to PGE1. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates. The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.

Inhibition of prostaglandin synthesis reduces cyclic AMP levels and inhibits chondrogenesis in cultured chick limb mesenchyme

The present study investigated effects of inhibiting the synthesis of prostaglandins (PGs) on cyclic AMP concentrations and chondrogenesis in cultured chick limb mesenchyme. Indomethacin produced concentration-dependent inhibition of both PGE(2) synthesis and chondrogenesis over a concentration range of 50--200 microM. Half maximal inhibition of PGE(2) was achieved with 50 microM concentrations of the drug which also produced visibly reduced amounts of cartilage matrix in cell cultures as evaluated by Alcian green staining on day 6 of culture. The inhibitory effects of indomethacin on chondrogenesis were largely reversed by addition of 1 mM dibutyryl cAMP, indicating that cells could still respond to cyclic AMP stimulation. Endogenous levels of cyclic AMP, which increased by 6 fold during the six days of culture in control cells, did not increase significantly from dissociated cells at the time of plating (day 0) in indomethacin- treated cultures. The results indicate that inhibition of the prechondrogenic rise in PGE(2) concentrations in limb mesenchyme prevents the increase in cyclic AMP levels which occur during this same period resulting in inhibition of chondrogenesis. The data provide further support for the hypothesis that PGE(2), through its effects on the adenylate cyclase-cAMP system, plays an important role in the differentiation of cartilage.

Influence of calcium, parathyroid hormone and ionophore a-23187 on cyclic nucleotide concentrations of isolated renal tubules

Abstract Effects of parathyroid hormone (PTH) and the divalent cation ionophore A-23187 on concentrations of cyclic AMP (cAMP) and cyclic GMP (cGMP) were investigated in isolated renal tubules from male hamsters. Both PTH and A-23187 increased cGMP concentrations, effects which required the presence of extracellular calcium. Increases in cGMP in response to both agents were proportionate to concentrations of calcium between 0.01 and 1.0 mM.Pretreatment of tubules with ionophore prevented any further increase in cGMP in response to PTH. Increases in cAMP in response to PTH were not altered by extracellular calcium, while smaller increases due to A-23187 were completely calcium-dependent. A-23187 increased efflux of 45Ca from renal tubules and resulted in decreased accumulation and net uptake of 45Ca at low extracellular calcium concentrations (2–3 μM). Increasing extracellular calcium (0.1–3 mM) proportionately reversed the inhibition in 45Ca accumulation and resulted in increases in cGMP levels which were directly related to increases in 45Ca accumulation. The results indicate that changes in cGMP concentrations of isolated renal tubules following either PTH or A-23187 are related to changes in intracellular calcium.

Responsiveness of adenylate cyclase to PGE2 and forskolin in isolated cells from micromass cultures of chick limb mesenchyme during chondrogenesis

Exogenous PGE2 stimulation of adenylate cyclase (AC) in intact and enzymatically dissociated micromass cultures of mesenchymal cells derived from the distal tip of stage 25 chick limb buds was examined over a six day period of culture. Responsiveness to PGE2 was measured in both dissociated and intact cell layers in an effort to determine if an inhibitory interaction occurred between PGE2 receptors and the extracellular matrix synthesized by differentiating chondrocytes. PGE2 responsiveness was maximal in both dissociated and intact prechondrogenic mesenchyme after 24 hours in culture and declined significantly as chondrocyte differentiation occurred on days 3 and 6. Equivalent activation of AC activity by PGE2 at each time point examined was noted in both cell groups. In contrast to the decreased responsiveness of differentiating chondrocytes to PGE2, stimulation of AC by forskolin resulted in increased levels of activity in differentiating chondrocytes of both cell groups between days 3-6. The results of the present study demonstrate that the decline in PGE2 responsiveness of differentiating chondrocytes most likely involves specific changes in the PGE2 receptor complex and not in either the interaction of the receptor with extracellular matrix components or a reduction in the available pool of AC present.

Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMP

Effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on chondrogenesis and concentrations of prostaglandin E2 (PGE2) and cyclic AMP (cAMP) were investigated in micromass cultures of chick limb mesenchyme derived from the distal tip of stage 25 limb buds. TPA completely inhibited chondrogenesis during the first 4 days of culture; however, a few small cartilage nodules formed by day 6. Relative to control cultures, both PGE2 and cAMP concentrations were altered by TPA treatment during the 6-day period of cell culture. Concentrations of both compounds increased in control cells during the first 24 h of culture and then declined during the remaining 5 days. In TPA-treated cells both PGE2 and cAMP levels increased progressively during the 6 days of days of cell culture, each being elevated at day 6 by twofold over control cells. The results suggest the presence of regulatory pathways important in chondrogenesis which occur independent of those initiated by PGE2 and the cAMP system.

Development of PTH-responsive adenylate cyclase activity during chondrogenesis in cultured mesenchyme from chick limb buds

SummaryThe present study investigated the development of parathyroid hormone (PTH)-responsive adenylate cyclase (AC) activity in chondrogenic cells differentiating from chick limb mesenchyme in culture. Mesenchyme from stage 25 chick embryos was removed from the distal tip (0.3 mm) of limb buds and cultured for a 6 day period in high density micromass cultures. Under these conditions, initial appearance of cartilage matrix and chondroblasts occurred on day 3 of culture and rapidly progressed over the next 3 days to produce, by day 6, a highly confluent and homogeneous layer of cartilage matrix and chondrocytes. Cells initially dissociated from limb mesenchyme on day 0 were essentially unresponsive to PTH, but development of AC-coupled, PTH receptors occurred rapidly during the initial 24 hours of culture. Based on data from dose-response experiments, prechondrogenic cells on day 1 of culture had synthesized their full complement of these receptors relative to fully differentiated chondrocytes in cultures at day 6. Inhibition of chondrocyte differentiation by retinoic acid did not significantly affect the initial development of AC-coupled, PTH receptors but it almost completely prevented synthesis of cartilage matrix. The results indicate that development of AC-coupled PTH receptors during chondrogenesis precedes, by at least 48 hours, overt differentiation of chondrocytes and the accumulation of cartilage-specific extracellular matrix and appears to represent one of the earliest reported events in chondrocyte differentiation. The lack of effect of retinoids on development of these receptors indicates that the inhibitory effects of retinoids on differentiating cartilage are at least somewhat specific for genes regulating synthesis of extracellular matrix molecules.