Accelerator mass spectrometry at Arizona: geochronology of the climate record and connections with the ocean.

There are many diverse uses of accelerator mass spectrometry (AMS). Carbon-14 studies at our laboratory include much research related to paleoclimate, both with 14C as a tracer of past changes in environmental conditions as observed in corals, marine sediments and many terrestrial records. Terrestrial records such as forest fires can also show the influence of oceanic oscillations, whether they are short-term such as ENSO, or on the millennial time scale. In tracer applications, we have developed the use of 129I as well as 14C as tracers for nuclear pollution studies around radioactive waste dump sites, in collaboration with IAEA. We discuss some applications carried out in Tucson for several of these fields and hope to give some idea of the breadth of these studies.

Accelerator mass spectrometry at Arizona: geochronology of the climatic record and connections with the ocean.

There are many diverse uses of accelerator mass spectrometry (AMS). 14C studies at our laboratory include much research related to paleoclimate, with 14C as a tracer of past changes in environmental conditions as observed in corals, marine sediments, and many terrestrial records. Terrestrial records can also show the influence of oceanic oscillations, whether they are short term, such as ENSO (El Niño/Southern Oscillation), or on the millennial time scale. In tracer applications, we have developed the use of 129I as well as 14C as tracers for nuclear pollution studies around radioactive waste dump sites, in collaboration with IAEA. We discuss some applications carried out in Tucson, AZ, for several of these fields and hope to give some idea of the breadth of these studies.

Extremely large variations of atmospheric 14C concentration during the last glacial period.

A long record of atmospheric 14C concentration, from 45 to 11 thousand years ago (ka), was obtained from a stalagmite with thermal-ionization mass-spectrometric 230Th and accelerator mass-spectrometric 14C measurements. This record reveals highly elevated Delta14C between 45 and 33 ka, portions of which may correlate with peaks in cosmogenic 36Cl and 10Be isotopes observed in polar ice cores. Superimposed on this broad peak of Delta14C are several rapid excursions, the largest of which occurs between 44.3 and 43.3 ka. Between 26 and 11 ka, atmospheric Delta14C decreased from approximately 700 to approximately 100 per mil, modulated by numerous minor excursions. Carbon cycle models suggest that the major features of this record cannot be produced with solar or terrestrial magnetic field modulation alone but also require substantial fluctuations in the carbon cycle.

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.

Comparative effects of cytosine arabinoside and a prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme.

The present study was designed to compare effects of an established inhibitor of cell proliferation and growth, cytosine arabinoside (Ara C), with that of a prostaglandin E2 (PGE2) antagonist, AH6809, on chondrogenesis in cultured mesenchyme derived from stage 25 chick limb buds. Continuous treatment of cell cultures with 10(-4) M AH6809 prevented completely the twofold increases in DNA content of control cultures which occurred between Day 1 and Day 5 of culture and also produced 90% inhibition of chondrogenesis occurring in control cultures during this same period. Treatment of cells with Ara C (0.1-0.5 microgram/ml) produced equivalent inhibition of DNA content during the same time period; however, chondrogenesis, as evaluated on Day 5 of cell culture, remained at approximately 90% of control cultures. These results indicate that the inhibitory effect of PGE2 receptor blockade on cell growth in these cultures cannot account for the potent inhibitory effects observed on differentiation of cartilage and provide further evidence in support of the notion that PGE2 plays an important initiating role in the process of chondrocyte differentiation within limb mesenchyme.

Effects of a putative prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme.

In the present study, we have examined the effects of a putative antagonist of prostaglandin E2 (PGE2), AH6809, on chondrogenesis in serum-free cultures of mesenchyme from distal tips of stage 25 chick limb buds in order to test the hypothesis that endogenous PGE2, through receptor-linked adenylate cyclase (AC), initiates differentiation of cartilage in limb mesenchyme. Daily addition of 10(-4) M concentrations of AH6809 produced marked inhibition of chondrogenesis over a 5-day period of cell culture as evaluated by Alcian green binding to cartilage matrix components. Inhibition of chondrogenesis by this compound was further shown to be reversible and treatment of cells with the antagonist limited to periods when chondrocytes had differentiated and were actively secreting cartilage-specific matrix components had little effect. Preincubation of control cells in 10(-4) M concentrations of AH6809 inhibited PGE2-induced activation of AC by greater than 80% without significant (P greater than .05) inhibition of basal activity by the antagonist. Responses to parathyroid hormone, which increased AC activity by 7-fold, and forskolin which increased AC activity by 23-fold in control cells, were also uninhibited by preincubation in AH6809. The results demonstrate that blockade of PGE2-AC linked receptors in prechondrogenic limb mesenchyme inhibits chondrogenesis supporting the hypothesis that endogenous PGE2 concentrations in undifferentiated limb mesenchyme play an initiating role in the differentiation of cartilage.

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

The 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.

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 AMP1.

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.

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.

Chondrogenesis in chick limb mesenchyme in vitro derived from distal limb bud tips: changes in cyclic AMP and in prostaglandin responsiveness.

Chondrogenesis was monitored in micromass cultures of mesenchymal cells derived from the distal tip of stage-25 chick limb buds over a 6-day period. Alcian green staining and immunofluorescent localization of cartilage-specific proteoglycans revealed the appearance of cartilage matrix by day 3 of cell culture. By day 6, cultures contained a uniform and homogeneous population of fully differentiated chondrocytes throughout the cell layer, with only a narrow rim of nonchondrogenic cells around the extreme periphery of the culture. Synthesis of sulfated glycosaminoglycans also progressively increased between days 3 and 6, being 8-fold higher at day 6 than at day 1 of culture. Both adenylate cyclase (AC) activity and cAMP concentrations increased dramatically during the first 2 days of culture, reaching maximal levels by day 2, which remained elevated and stable throughout the remaining chondrogenic period (days 3-6). Responsiveness of both AC and cAMP concentrations of the cells to PGE2 was maximal by day 1 of culture and was increased over control cells by 12-fold and 8-fold respectively. Both responses, however, were dramatically reduced by day 3, at which time the initiation of cartilage formation was apparent. Responsiveness of cells during the prechondrogenic period to PGE2 was relatively specific in that no effects could be demonstrated with equivalent concentrations of PGF2 alpha or 6-keto-PGF1 alpha, although PGl2 did produce increases in cAMP concentrations of about 50% of those of PGE2. These results indicate that previously reported changes in the cAMP system in heterogeneous cell cultures derived from whole limb buds reflect changes occurring in the chondrogenic cell type and indicate further that peak responsiveness of the cAMP system of these cells to prostaglandins is restricted to prechondrogenic developmental periods.

Limb development in chick embryos: cyclic AMP-dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis.

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20-23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25-28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP-dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80-85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane-enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP-dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP-protein phosphorylation system in the differentiation of limb tissues.

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.

The morphology and hormonal responsiveness of developing skeletal elements in chick limb buds.

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.

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.

Enhancement by ethanol of parathyroid-hormone-stimulated cyclic AMP accumulation in isolated renal tubules.

The effects of ethanol on parathyroid hormone (PTH)-induced increases in adenosine 3':5'-phosphate (cAMP) concentrations were studied in renal cortical tubules of hamsters in vitro. Ethanol concentrations between 0.1 and 3% were found to augment the PTH response in a dose-related way while, concentrations greater than 3% produced a dose-related inhibition of the PTH response. In the absence of PTH, ethanol did not significantly elevate cAMP accumulations at any concentrations tested. In contrast to its effect on intact tubule cells, ethanol did not alter either adenylate cyclase or phosphodiesterase in renal cortical homogenates. Indomethacin, however, produced a concentration-related inhibition of the ethanol-potentiated response without altering the effects of PTH alone. The results suggest a possible involvement of prostaglandins in the potentiating effect of ethanol on the PTH-dependent accumulation of cAMP in renal tubules.

Lymphocyte cyclic AMP levels antepartum and postpartum as an indirect assessment of the immune system during pregnancy.

The literature dealing with the immunological state of the pregnant woman has been conflicting. The concentrations and activity of a number of hormones and proteins which modify lymphocytic activity have been measured both in vivo and in vitro during pregnancy. Most of the differences between reported studies can be reconciled to technical or experimental variations. In some instances, the purported suppressive effects of embryonic proteins such as HCG have actually been caused by the impurity of the preparation studied. We have attempted to approach the question of whether or not the pregnant woman is immunosuppressed by studying a regulatory material in the lymphocytes. It is known that cAMP is a mirror of lymphocytic activity and that low levels of cAMP may indicate a high degree of reactivity, while high levels are present when lymphocyte reactivity is low. In an initial study, ten women volunteered to have blood drawn in the last trimester and two months postpartum. Cyclic AMP was extracted from lymphocyte-enriched leukocytes and stored until all samples were available from all patients so that the analysis could be made simultaneously. Five samples were obtained from healthy nonpregnant women in the same age range. Postpartum and nonpregnant women were found to have significantly elevated levels of cAMP as compared to the lymphocytes obtained in the third trimester of pregnancy. The experiments were then repeated using seven more patients. The same significant increase in postpartum lymphocyte cAMP concentrations were found. The precise reason(s) for this is not known, but may be due to increased suppressor cell activity.

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation.

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.

Interactions between parathyroid hormone and prostaglandins on renal cortical cyclic AMP.

Effects of parathyroid hormone (PTH) and several prostaglandins (PGs) on cyclic AMP (cAMP) metabolism were studied and compared in isolated renal cortical tubules from male hamsters. Both production and intracellular degradation of cAMP were increased by PTH and each of the PGs tested (PGE2, PGE1, PGI2). Production of cAMP was increased to similar levels by maximal concentrations of PTH and each PG, however, degradation of cAMP was significantly higher in response to PTH than with any of the PGs. This difference in intracellular degradation of cAMP was responsible for the much higher concentrations of cAMP in renal cortical tubules exposed to PGs (PGE1, PGE2, PGI2) than to PTH. Submaximal amounts of each PG produced additive increases in cAMP concentrations in the presence of maximal amounts of PTH. Additivity of the combined responses was lost, however, as the PGs concentrations reached their maxima. The results suggest that renal PGs (PGE2 and PGI2) may modulate the effects of PTH on cAMP concentrations in renal cortical tubules.

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

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.