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.     

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.     

Effect of metabolic inhibitors and temperature on uptake and translocation of ca and k by intact bean plants

The dependence of Ca uptake and translocation by intact roots of Phaseolus vulgaris on concurrent root metabolism was investigated using ⁴⁵Ca-labeled Hoagland solutions at one-half and one-twentieth strength (2.5 and 0.25 mM Ca²⁺). Adsorbed and absorbed ⁴⁵Ca fractions in the roots were distinguished on the basis of the time course of exchange with the outer solution. Uptake of ⁴²K, of which the characteristics are better known, was measured for comparison. The absorbed ⁴⁵Ca fraction showed a markedly nonlinear increase with time in contrast to the near linear increase in ⁴²K. Exposure of roots to cyanide, arsenate, 2,4-dinitrophenol, or low temperatures caused only slight reductions in ⁴⁵Ca absorption by roots, but significant reductions of ⁴²K. In all treatments involving inhibitors and low temperatures, the translocation to shoots of both ⁴⁵Ca and ⁴²K was strongly inhibited. The conclusion that much of the absorbed ⁴⁵Ca fraction in the root tissue is taken up by processes which are not rate-limited by metabolism is discussed.     

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.     

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.