Adenosine 5'-triphosphate synthesis and metabolism localized in neurites of cultured sympathetic neurons

Adenosine triphosphate synthesis and metabolism in cultured sympathetic neurons was studied after the incorporation of [2-3H]adenine into intact or microdissected neurites to determine whether ATP is provided locally during neurite outgrowth, when and where it is synthesized and how its levels are regulated at rest and following depolarization. Neurites maintained an independent capability for synthesis of ATP at any stage of growth: [3H]ATP levels increased in neurites in direct proportion to neurite length and equivalent amounts of [3H]ATP were synthesized by intact neurites, by neurites separated from cell bodies or by neurites further segmented into sections. Thus, metabolic labelling of cultured neurons with [3H]adenine provides a simple method to measure relative neurite outgrowth. Neurite ATP was maintained mainly by respiration but also by glycolysis and [3H]ATP levels were stable for at least 14 h after adenine withdrawal when cells were at rest. Depolarization overcame respiratory control, causing a quantitative conversion of ATP to adenosine monophosphate (AMP) and inosine monophosphate (IMP) and the release of nucleosides (adenosine and inosine) and nucleotides [adenosine diphosphate (ADP) and adenosine monophosphate (AMP)]. Release of nucleosides, but not of nucleotides or [3H]noradrenaline, was enhanced by NaN3 or 2-deoxyglucose under nondepolarizing conditions and was prevented by the adenosine transport inhibitor p-nitrobenzyl-6-thioinosine. It is concluded that neurites can use local mechanisms for ATP synthesis that do not depend on a functional connection to the cell body. Any metabolic stress which causes ATP breakdown causes these cells to express a transient purinergic phenotype involving release of adenosine and inosine by facilitated diffusion. To promote the release of purine nucleotides, however, more specific stimuli are required.     

Adenosine is the primary precursor of all purine nucleotides in Trichomonas vaginalis

Trichomonas vaginalis, a parasitic protozoan and the causative agent of trichomoniasis, lacks de novo purine nucleotide synthesis and possesses a unique purine salvage pathway, consisting of a bacterial type purine nucleoside phosphorylase and a purine nucleoside kinase. It is generally believed that adenine and guanine are converted to their corresponding nucleosides and then further phosphorylated to form AMP and GMP, respectively, as the main as well as the essential pathway of replenishing the purine nucleotide pool in the organism. Formycin A, an analogue of adenosine, inhibits both enzymes as well as the in vitro growth of T. vaginalis with an estimated IC(50) of 0.27 microM. This growth inhibition was reversed by adding adenine to the culture medium but not by adding guanine or hypoxanthine. Furthermore, T. vaginalis can grow in semi-defined medium supplemented with only adenine but not with guanine or hypoxanthine. Radiolabeling experiments followed by HPLC analysis of the purine nucleotide pool in T. vaginalis demonstrated incorporation of [8-14C]adenine into both adenine and guanine nucleotides, whereas [8-14C]guanine was incorporated only into guanine nucleotides. Substantial adenosine deaminase activity and significant IMP dehydrogenase and GMP synthetase activities were identified in T. vaginalis lysate, suggesting a pathway capable of converting adenine to GMP via adenosine. This purine salvage scheme depicts adenosine the primary precursor of the entire purine nucleotide pool in T. vaginalis and the purine nucleoside kinase one of the most pivotal enzymes in purine salvage and a potential target for anti-trichomoniasis chemotherapy.     

Effect of dipyridamole on adenosine incorporation into hypoxanthine nucleotides of fresh human red cells

Fresh human red cells were incubated for 2 hours in a medium containing adenosine, pyruvate and inorganic phosphate (APP medium), or in APP medium supplemented with 10(-4) M dipyridamole (APPD medium). No measureable amount of ITP was found in fresh red cells, and the average IMP content in these cells was 0.18 +/- 0.09 mumol/g Hb. After 2 hours incubation in APP medium, the IMP content increased almost 8.5-fold to 1.52 +/- 0.78 mumol/g Hb. Under these conditions the ITP level also increased to 1.40 +/- 0.84 mumol/g Hb. After 2 hours incubation of red cells in APPD medium, the average IMP content increased to 5.30 +/- 2.33 mumol/g Hb, about 3.5 times that found in APP medium. At the same time ITP content was about 53.6% lower, that is 0.65 mumol/g Hb. In red cells incubated in APPD medium, penetration of 8-14C-adenosine decreased by 50%, and incorporation of this nucleotide into the pool of all free nucleotides also decreased by 18.2% as compared to red cells incubated in APP medium. It is concluded that IMP is probably formed directly from AMP gained by the phosphorylation of adenosine during its penetration.     

Oxidized and ubiquitinated proteins may predict recovery of postischemic cardiac function: essential role of the proteasome

This study examined the hypothesis that postischemic levels of oxidized and/or ubiquitinated proteins may be predictive of functional recovery as they may be indicative of activity of the 20S and/or 26S proteasomes, respectively. Subjecting isolated rat hearts to 15 min of ischemia had no effect on 20S- and 26S-proteasome activities; however, both were significantly (p < 0.05) decreased by 70% and 54%, respectively, following 30 min of ischemia and 60 min of reperfusion, changes associated with increased levels of protein carbonyls and ubiquitinated proteins. Preischemic treatment of hearts with the proteasome inhibitor, MG132, resulted in dose-dependent decreases (p < 0.05) in recovery of postischemic function [MG132 (microM), heart rate x pressure product: 0, 11,158 +/- 2,423; 6, 11,400 +/- 3,009; 12, 5,513 +/- 2,225; 25, 2,325 +/- 992] and increased accumulation of ubiquitinated proteins. Preconditioning with repetitive ischemia (IP) or preischemic treatment with nicorandil (Nic) resulted in a significant increase in postischemic 20S-proteasome activity after 60 min of reperfusion (control, 95 +/- 4; IP, 301 +/- 65; Nic, 242 +/- 61 fluorescence units). Only Nic had similar effects on 26S-proteasome activity. These results support the conclusion that a correlation exists between eventual recovery of postischemic function and levels of oxidized and/or ubiquitinated proteins, a phenomenon that may be dependent on activity of the 20S and 26S proteasomes.     

Adenosine and its nucleotides stimulate proliferation of chick astrocytes and human astrocytoma cells.

Aqueous extracts of the brains of 18-day-old white Leghorn chicken embryos contain several substances that stimulate proliferation of primary cultures of chick brain astrocytes. Most of the mitogens are peptides. Purification of one mitogenic fraction was obtained by centrifugation, passage through Amicon Diaflo membranes of nominal molecular weight cutoffs 30, 1 and 0.5 kDa, ion exchange chromatography and reverse phase high performance liquid chromatography (HPLC) using a Deltapak C18 column. The mitogenic fraction contained no amino acids. On the basis of its behaviour on thin layer chromatography, its ultraviolet absorption spectrum, its 1H and 31P nuclear magnetic resonance spectra and its behaviour on positive and negative ion fast atom bombardment mass spectrometry, the mitogenic material was identified as adenosine-5'-monophosphate (AMP). Other adenosine compounds including adenosine, ADP and ATP also stimulated proliferation of and [3H]leucine incorporation into primary cultures of astrocytes. Nitrobenzylthyioinosine (NBTI), an inhibitor of nucleoside transport, did not prevent the stimulation of [3H]leucine incorporation into cultured astrocytes. Polyadenylic acid (Poly A), that mimics the effect of adenosine at adenosine receptors, also stimulated proliferation of the astrocytes. The effects of adenosine and Poly A were not inhibited by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) but were inhibited by 1,3-dipropyl-7-methyl-xanthine (DPMX), indicating that adenosine and Poly A acted at the cell surface, likely through adenosine A2 receptors. The stimulatory effect of ATP was biphasic. The proliferative effect of low, but not of high, concentrations of ATP were abolished by DPMX. The purinergic P2 receptor agonist 2-methylthioATP and, at higher concentrations, the P2y agonist, alpha,beta-methyleneATP also stimulated incorporation of [3H]thymidine. These data indicate that high concentrations of ATP stimulate cell proliferation through at a P2, possibly a P2y receptor. These results have considerable biological significance. After brain injury, or when cells in brain die or become hypoxic, nucleotides and nucleosides are released from the cells. Their extracellular concentrations can exceed those required to stimulate astrocyte proliferation in vitro. Therefore they may be partly responsible for gliotic changes following cell death in brain.     

Distribution of adenine nucleotides in the perfused rat heart.

A computer technique for determination of the distribution of adenine nucleotides among compartmented, protonated, and metal-chelated species has been developed for the perfused rat heart. This procedure requires knowledge of tissue levels of creatine, creatine phosphate, ATP, ADP, and AMP and the glycolytic and respiration rates. The method is applicable to any physiological state of the organ and has been applied to transient behavior in aerobic, anoxic, and ischemic hearts. The results suggest that ADP uptake and ATP export by mitochondria are normally linked and equal in rate during aerobic metabolism or short-term anoxia but become separate and unequal during ischemia, so that mitochondrial adenine nucleotides, primarily AMP, accumulate.     

Neuromodulation by adenine nucleotides, as indicated by experiments with inhibitors of nucleotide inactivation

The adenine nucleotides AMP, ADP and ATP (3 X 10(-7) M and above) inhibited contractile responses to transmural nerve stimulation in guinea-pig ileum longitudinal muscle via a prejunctional action. Nucleotides assumed to inhibit the degradation of adenine nucleotides were employed to determine whether inhibition of contractile responses was elicited by adenine nucleotides per se, or required breakdown to adenosine. The IMP or 2'-deoxy AMP enhanced the prejunctional inhibitory effect elicited by AMP. A similar enhancement of the inhibitory effect of ADP and ATP was seen after administration of IDP and ITP, respectively. The inhibitory effect of adenosine was not enhanced by inosine, IMP or IDP. The 5'-nucleotidase inhibitor, TDP enhanced inhibition elicited by ADP. In contrast, alpha, beta-meADP did not influence the prejunctional inhibitory effect elicited by the adenine nucleotides. However, the combination of alpha, beta-meADP and IMP enhanced the inhibitory effect of ATP. The postjunctional contractile effect elicited by ADP and ATP was enhanced by pretreatment with inosine nucleotides, alpha, beta-meADP or TDP, indicating decreased inactivation of ADP and ATP during concurrent nucleotide administration. The fact that the prejunctional effect of adenine nucleotides can be enhanced by forms of pretreatment known to antagonize the breakdown of adenine nucleotides, constitutes strong evidence for prejunctional action per se by adenine nucleotides.     

Fatty acid synthesis in the arrested rabbit heart during ischemia

In rabbit hearts arrested by a carnosine-buffered cardioplegic solution the incorporation of [1-¹⁴C]acetate into lipids was investigated. After 10–20 and 60 min of ischemia the radioactivities of phospholipids, mono-, di-, and triacylglycerols, acyl-CoA, acylcarnitine, and free fatty acids were determined. In the first period of ischemia mainly acylcarnitine was labelled (ca. 50%) and only 25% of [¹⁴C]-activity was found in phospholipids which showed the lowest specific activity of all lipid classes. After 60 min of ischemia the percentage of total radioactivity of acylcarnitine and phospholipids was decreased, whereas that of neutral lipids was increased to more than 50%. During the increase of total radioactivity the relative specific activity of all lipids decreased except that of triacylglycerols. Only fatty acids up to chain lengths of 16 carbon atoms were labelled. Lauric and myristic acid had high specific activities. These results indicated de novo synthesis of fatty acids accumulating in triacylglycerols during ischemia of the arrested heart.     

Independence of onset of compensatory kidney growth from changes in renal function.

Renal function was measure before and shortly after uninephrectomy in mice to evaluate if work expended in the reabsorption of glomerular filtrate plays a role in the initiation of compensatory growth. To exclude the possibility of small but undetectable increments in glomerular filtration rate and absolute sodium reabsorption these functions were experimentally reduced immediately after uninephrectomy and sham nephrectomy. The onset of growth was indicated by an increased rate of [14C]choline incorporation into phospholipid in renal cortical slices. [14C]choline incorporation increased significantly only after uninephrectomy and remained unchanged after sham operation regardless of the magnitude or direction of the concurrent change in sodium reabsorption. The rate of incorporation increased by 40 +/- 8% (P less than 0.005) in uninephrectomized animals whose sodium reabsorption was reduced by 34 +/- 6% (P less than 0.001) and rose 45 +/- 11% (P less than 0.005) when sodium reabsorption remained unchanged. These results indicate that compensatory kidney growth is not triggered by an increase in renal work expended in the reabsorption of glomerular filtrate; in fact, it can occur when reabsorptive work is substantially decreased.     

Succinate-dependent energy generation in Ascaris suum mitochondria

Phosphorylation in isolated Ascaris suum mitochondria was much greater in the presence of malate than succinate, but, in the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochondrial ATP levels. Succinate-dependent phosphorylation was stimulated aerobically and this stimulation was due almost entirely to a site I, rotenone-sensitive, phosphorylation. Increased substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, 14CO2 evolution from 1,4-[14C]succinate was stimulated and NADH/NAD+ ratios were elevated, but the formation of [14C]propionate was unchanged. It appears that succinate was metabolized to pyruvate and acetate, and NADH, generated from the decarboxylations of malate and pyruvate, was the primary source of reducing power fueling electron-transport. The terminal oxidase and final electron-acceptor are still not clearly defined. However, ferricyanide, H2O2, and 100% oxygen all stimulated succinate-dependent phosphorylation. A possible role for cytochrome c peroxidase in A. suum mitochondrial metabolism is discussed.     

Adenosine-5'-O-(3-thiotriphosphate) binding to human neutrophils. Evidence for a common nucleotide receptor.

Human polymorphonuclear neutrophils (PMN) respond to ATP with an elevation in intracellular calcium and a marked enhancement of O2-production in response to stimulation by the chemotactic peptide N'-formyl-Met-Leu-Phe (FMLP). These pertussis toxin-sensitive pathways appear to be mediated by a nucleotide receptor(s) on the surface of human PMN. In the current study, we have examined the binding to intact human PMN of the ATP analog, adenosine 5'-O-(3-thio[35S] triphosphate) [( 35S]ATP gamma S). On the basis of Scatchard analysis, the binding of [35S]ATP gamma S involves at least two sites, one of high and one of low affinity. In the presence of sodium thiophosphate, a compound which did not affect intracellular increases in calcium induced by ATP or N'-formyl-Met-Leu-Phe, a significant fraction of the [35S]ATP gamma S binding was eliminated. This reduction involved both high and low affinity binding of [35S]ATP gamma S and was related to a reduction in numbers of binding sites. The Kd values for the high affinity binding site were unaffected by the presence of sodium thiophosphate, although the low affinity Kd values were numerically increased by 2-fold. In the presence of thiophosphate, [35S]ATP gamma S binding was specific, saturable, and reversible, and was related to a single class of high affinity (Kd = 36 +/- 19 nM) binding sites (184 +/- 144 sites/cell), together with a second class of low affinity (Kd = 1110 +/- 503 nM) binding sites (13,562 +/- 6,851 sites/cells). Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block [35S]ATP gamma S binding to PMN, revealed a rank order of ATP gamma S greater than ATP greater than 2-MeS-ATP = 8-Bromo ATP greater than ADP = ITP greater than AMP-PCP = GTP much greater than CTP. A comparison between the ability of nucleotides to compete with [35S]ATP gamma S binding and their ability to induce a biologic response (elevation of intracellular calcium) revealed a close correlation (r2 = 0.83). These findings support the possibility of a common nucleotide PMN receptor functionally linked to a cellular response which involves increases in intracellular calcium.     

Coronary autoregulation and purine release in normoxic heart at various cytoplasmic phosphorylation potentials: disparate effects of adenosine

The impacts of energy-yielding substrates on coronary flow autoregulation, cytoplasmic phosphorylation potential {[ATP]/([ADP][P_i])} and purine nucleoside production were studied in Langendorff-perfused guinea pig hearts. The perfusion medium was substrate-free or contained glucose alone or in combination with pyruvate, lactate, acetate, or octanoate as fatty acid. When coronary flow was adjusted for myocardial oxygen consumption, only pyruvate supported near-perfect intrinsic autoregulation at highly sustained [ATP]/([ADP][P_i]) and low interstitial adenosine concentrations ([Ado]). In contrast, hearts perfused with substrate-free medium were deenergized at very high [Ado], especially at supraphysiological pressures, which markedly impaired autoregulatory vasoconstriction. Thus, efficient autoregulatory vasoconstriction was associated with high [ATP]/ ([ADP][P_i]) at low [Ado]. On the other hand, autoregulatory vasodilation at subphysiological pressures was associated with increased [Ado] and partially blocked by 28 M theophylline demonstrating (partial) adenosine mediation. Massive accumulation of IMP, especially relative to free cytoplasmic AMP, occurred at normal intracellular pH during myocyte deenergization by substratefree perfusion. This may indicate allosteric activation of native AMP deaminase in situ, perhaps because of collapse of [ATP]/([ADP][P_i]). Similarly, rates of adenosine plus inosine release and of total purines, also including urate, exhibited non-linear sigmoidal rather than linear or rectangular hyperbolic dependences on free cytoplasmic AMP concentration (not total AMP content). Since inclusion of IMP as a co-variable of free AMP appreciably improved the sigmoidal fits, IMP appeared to be a significant precursor of released inosine in guinea pig heart.     

Does resveratrol induce pharmacological preconditioning?

Resveratrol is a grape component with complex pharmacology related to its antioxidant activity. Little is known about the direct effects of resveratrol on the myocardium. We tested whether resveratrol administration before ischemia could attenuate ischemic/reperfusion damage. We examined how resveratrol affects high-energy phosphate metabolism (31P-nuclear magnetic resonance) and contractility of isolated Langendorff perfused rat hearts subjected to 20 min no-flow ischemia and 30 min reperfusion. During 10 min resveratrol infusion (10 microM) before ischemia, basal phosphorylation potential dropped by 40% (p < 0.05 vs. preinfusion value) without affecting contractility. The level of effluent adenosine was increased by 68%, parallel to a 50% increase in coronary flow. Resveratrol significantly improved postischemic recovery of rate-pressure product (62 +/- 5.2 vs. 23 +/- 8.1% of controls; p < 0.05). The metabolic pattern following resveratrol infusion was similar to that produced by ischemic preconditioning, suggesting that an increase in adenosine availability is involved in cardioprotection.     

Improved high-performance liquid chromatographic assay for the determination of "high-energy" phosphates in mammalian skeletal muscle. Application to a single-fibre study in man.

A sensitive and reproducible method for the determination of adenine nucleotides (ATP, IMP) and creatine compounds [creatine (Cr), phosphocreatine (PCr)] in freeze-dried single human muscle fibre fragments is presented. The method uses isocratic reversed-phase high-performance liquid chromatography of methanol extracts. Average retention times (min) of ATP, IMP and PCr, Cr from standard solutions were 10.6+/-0.42, 2.11+/-0.06 (n=6) and 10.5+/-0.31 and 1.19+/-0.02 (n=9), respectively. Detection limits in extracts from muscle fibre fragments were 2.0, 1.0, 3.0 and 2.0 mmol/kg dm, respectively. The assay was found successful for analysis of fibre-fragments weighing > or = 1 microg.     

Enhanced recovery of renal ATP with postischemic infusion of ATP-MgCl2 determined by 31P-NMR

The postischemic infusion of ATP-MgCl2 will enhance the recovery of both glomerular and tubular function. To assess the effect of ATP-MgCl2 on tissue nucleotides, 31P nuclear magnetic resonance (31P-NMR) spectra were obtained continuously in vivo from the left kidney of rats that had been subjected to 45 min of renal ischemia and then infused with either normal saline or ATP-MgCl2. 31P-NMR spectra with distinct peaks for alpha-, beta-, and gamma-phosphate of ATP, sugar phosphate, and inorganic phosphate were collected every 7 min before, during, and after renal artery occlusion. During ischemia, the ATP beta-peak (the only peak unique to ATP) fell rapidly to 10% of control values in both groups of animals. By 120 min after the ischemic insult, the animals treated with ATP-MgCl2 had recovery of renal ATP to 89 +/- 2.6%, which is significantly greater (P less than 0.001) than 65.2 +/- 1.8% found in rats given normal saline. These data indicate that 1) 31P-NMR can be used to assess renal ATP levels continuously in vivo; 2) during renal ischemia ATP levels fall quickly to less than 10% of control values; 3) tissue ATP returns relatively slowly to control values in rats given normal saline, whereas postischemic treatment with ATP-MgCl2 results in an accelerated recovery of tissue ATP levels. These findings provide a biochemical correlate to the improvement in renal function previously described.     

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23).     

Some aspects of adenosine triphosphate synthesis from adenine and adenosine in human red blood cells

1. The synthesis of ATP has been studied in human erythrocytes. Fresh cells showed no net synthesis of ATP when incubated with adenine or adenosine, although labelled adenine was incorporated into ATP in small amounts.2. Cold-stored cells (3-6 weeks old) became progressively depleted of adenine nucleotides but incubation with adenosine or adenine plus inosine restored the ATP concentration to normal within 4 hr. Incorporation of labelled adenine or adenosine into the ATP of incubated stored cells corresponded to net ATP synthesis by these cells.3. Synthesis of ATP from adenosine plus adenine together was 75% derived from adenine and only 25% from adenosine, indicating that nucleotide synthesis from adenine inhibits the simultaneous synthesis of nucleotide from adenosine.     

5'-triphosphate recycles independently of acetylcholine in cholinergic synaptic vesicles.

The effect of hemicholinium-3 (HC-3) on vesicular contents in acetylcholine (ACh) and 5-triphosphate (ATP) and the vesicular incorporation of 14C-label derived from [14C]choline ,nd 3H-label derived from [3H]adenosine was investigated after low frequency stimulation (with a subsequent rest period) of the Torpedo electric organ. HC-3 (100 microM) caused an increased depletion of vesicular ACh and blocked the incorporation of 14C-label whereas contents in vesicular ATP and 3H-incorporation were identical with and without HC-3. HC-3 also blocked the recovery of electrical response of the tissue after stimulation but did not cause a change in vesicle numbers. The result suggest that synaptic vesicles continue to recycle ATP in the absence of recycling of ACh and that vesicular uptake and storage of the two components are not coupled to each other.     

Regulation of chondrosarcoma metabolism by cyclic adenosine 3'5'-monophosphate

The in vitro effects of cyclic AMP on amino acid transport and synthesis of macromolecules in the Swarm rat chondrosarcoma were investigated using the cyclic AMP analogue, N6-monobutyryl cyclic AMP (MBcAMP), and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). Amino acid transport was assessed by measuring alpha-amino-[1-14C]isobutyrate (AIB) uptake. The synthesis of macromolecules was estimated by measuring radiolabeled precursor incorporation into total proteins, proteoglycan, and RNA. MBcAMP stimulated [14C]AIB uptake, [3H]uridine transport, and UTP formation. MBcAMP inhibited 35SO4 and [3H]leucine incorporation into proteoglycan and stimulated [3H]uridine incorporation into RNA. MIX elevated endogenous cyclic AMP levels in the Swarm rat chondrosarcoma and mimicked the effects of MBcAMP on AIB transport and radiolabeled precursor incorporation into macromolecules. For comparative purposes, the effects of MBcAMP on AIB uptake and macromolecule synthesis in rat costal cartilage segments were investigated. MBcAMP and MIX stimulated AIB uptake by costal cartilage segments, inhibited [3H]leucine incorporation into total protein and 35SO4 incorporation into proteoglycan, and had no effect on [3H]uridine incorporation into RNA.