Evidence for negative control of growth by adenosine in the mammalian embryo: induction of Hmx/+ mutant limb outgrowth by adenosine deaminase.

Abstract

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)

• Authors: T B Knudsen, W A Elmer
• Journal: Differentiation; research in biological diversity (Differentiation) Vol. 33 Issue 3 Pg. 270-9 ( 1987) ISSN: 0301-4681 [Print] England
• PMID: 3596088 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Phenylisopropyladenosine
• Nucleoside Deaminases
• Adenosine Deaminase
• Adenosine

Topics

• Adenosine (analogs & derivatives, physiology)
• Adenosine Deaminase (metabolism)
• Animals
• DNA Replication
• Embryo, Mammalian (physiology)
• Embryonic and Fetal Development (drug effects)
• Female
• Hindlimb (embryology)
• Homeostasis
• Mice
• Mice, Mutant Strains
• Mutation
• Nucleoside Deaminases (metabolism)
• Organ Culture Techniques
• Phenylisopropyladenosine (pharmacology)
• Pregnancy