The purpose of the present study was to classify adenosine receptors into A1 and A2 subtypes in a wide range of isolated tissues and cell types (rat adipocytes and atria, guinea‐pig ileum and atria (A1); guinea‐pig aorta, dog coronary artery and human platelets and neutrophils (A2)) using the R‐ and S‐diastereoisomers of N‐phenylisopropyladenosine (PIA), N‐cyclopentyladenosine (CPA), the novel compound, N‐[(1S,trans)‐2‐hydroxycyclopentyl]adenosine (GR79236), N‐[(2‐methylphenyl)methyl]adenosine (metrifudil), 2‐(phenylamino)adenosine (CV1808), and 2‐[[2‐[4‐(2‐carboxyethyl)phenyl]ethyl]amino]‐N‐ethylcarboxamidoadenosine (CGS21680); N‐ethylcarboxamidoadenosine (NECA) was used as a standard. Results obtained in all tissue preparations previously reported to contain A1‐receptors could be described by a single rank order of agonist potency: CPA ≥ GR79236, R‐PIA ≥ NEC A > > S‐PIA ≥ metrifudil ≥ CV1808, CGS21680. In contrast, two distinct rank orders of agonist potency were observed in preparations previously reported to contain A2‐receptors. In dog coronary artery, human neutrophils and platelets the rank order of potency was: CV1808, CGS21680 ≥ NECA > R‐PIA ≥ metrifudil ≥ CPA > GR79236, S‐PIA. However, in guinea‐pig aorta the rank order was: NECA > metrifudil > R‐PIA, CPA > CV1808, GR79236 ≥ S‐PIA, CGS21680. The results of this study are consistent with the existence of three types of adenosine receptor: A1‐and two subtypes of A2‐receptor. The receptor present in dog coronary artery, human platelets and neutrophils, probably corresponds to the A2a subtype, whilst that present in the guinea‐pig aorta may be of the A2b subtype.
IN connection with work on the isotope uptake by normal and malignailt cells, the synthesis of desoxyribose nucleic acid (DNA) by normal and leukaemic bone marrow and blood cells was investigated. This paper reports the results obtained on the incorporation of 32P orthophosphate and adenine-8_14C into DNA by human bone marrow cells in vitro, and the effect of a single dose of 5000 r X-ray irradiation on DNA synthesis. METHODS.The technique for culturing human bone marrow in vitro has been described in detail elsewhere (Lajtha, 1952a). Cell suspensions were cultured in a liquid medium and the isotope was added after the cultures were set up. At the end of the culture period (3-26 hours) smears were made from the cultures and stained autoradiographs were prepared for differential counts and grain counting, using a hioh resolution stripp'mg film technique (Laitha, 1952b(Laitha, , 1954a In nearly aR experiments described here the isotope was a'dded so as to have a concentration of 5 Itc. /ml. niedium ; 32P orthophosphate (as supplied by A. E.R. E., Harwell, in a pH 7 phosphate buffer) was used. Lower concentrations of 32P failed to give satisfactory autoradiographs. In a few experiments 10 /tc./ml. mediUM 32P was used, but the general background on the autoradiographs was 14C then rather high due to scatter and cross fire. The concentration of adenine-8_ in all experiments was 0-5 /tc./ml. medium. The specific activity of the adenine 14C sulphate, as supphed from the Radiochemical Centre, Amersham, was 10 PC-1mg-All smears were alcohol fixed, and, to obtain autoradiographs from DNA only, one set of smears from each culture bottle was treated with N HCI at 60' C. for 6. minutes. This degree'of hydrolysis was found to remove aR non-DNA phosphorus as weR as all non-DNA adenine from the cells, while leaving DNA phosphorus and DNA adenine in situ, thus obviating the necessity for the use of the einzyme ribonuclease (Lajtha, 1954b). Adenine 14C administered to cells appears both as labelled adenine and labelled guanine in the DNA (Hamilton, 1953). No attempts to separate labelled adenine from labeHed guanine have been made in the experiments ; the activity of the DNA has been measured as a whole. The autoradiographs were exposed for 10-14 days. AR autoradiographs belonging to the same experiment were coated with the same batch of stripping film, exposed the same length of time at the same temperature (3-4' C.), and developed in one batch of developer simultaneously, and, finally, were stained simul-
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