Evidence for negative control of growth by adenosine in the mammalian embryo: induction of Hmx/+ mutant limb outgrowth by adenosine deaminase.
We investigated the growth-regulatory actions of adenosine and adenosine deaminase (ADA) during embryonic limb development in the mouse. Polydactylous outgrowth, an expression of the Hemimelia-extra toe (Hmx/+) mutant phenotype, was experimentally regulated in hindlimb buds explanted into a serum-free in vitro system at stage 18 of gestation. Its expression was promoted by exposure to 0.1 or 0.2 IU/ml exogenous ADA and suppressed by co-exposure to 10 nM (-)-N6-(R-phenylisopropyl)-adenosine (N6-PIA). Evidence that N6-PIA acted as a high-affinity agonist against the external adenosine receptor was provided by experiments in which 100 microM caffeine, a known antagonist, competitively blocked its effect. The endogenous adenosine content was analyzed by reversed-phase high-performance liquid chromatography with fluorometric detection following its conversion to the 1,N6-ethenoadenosine derivative. At stage 18, the adenosine levels were 0.5 pmol/micrograms DNA in whole embryos and 0.08 pmol/micrograms DNA in hindlimb buds. At the same stage, partially purified extracts of the embryonal plasma enriched fraction contained high levels of ADA activity (0.04-0.06 IU/embryo, or 0.7-1.0 IU/mg protein). In contrast, blood cells contained 0.0001 IU/embryo (or 0.01 IU/mg protein). This enzyme occurred as a single kinetic form with a molecular weight of 45000-47000 daltons and an apparent Km of 36-38 microM. Its presence in the embryonal plasma argues against an endocrine mechanism of adenosine secretion in favor of autocrine (self-regulatory) or paracrine (proximate-regulatory) mechanisms. Taken together, our results suggest that the in vitro outgrowth of the prospective polydactylous region is induced upon escape from the local growth-inhibitory influence of extracellular adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)
D000241:Adenosine / Q000031:analogs & derivatives* / Q000502:physiology*; D000243:Adenosine Deaminase / Q000378:metabolism*; D000818:Animals; D004261:DNA Replication; D004622:Embryo, Mammalian / Q000502:physiology; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D006614:Hindlimb / Q000196:embryology; D006706:Homeostasis; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D009700:Nucleoside Deaminases / Q000378:metabolism*; D009924:Organ Culture Techniques; D010660:Phenylisopropyladenosine / Q000494:pharmacology*; D011247:Pregnancy
D010660:Phenylisopropyladenosine; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase; D000241:Adenosine