We demonstrated the distribution pattern of ecto-5'-nucleotidase (5'-Nu) in rat kidney by enzymatic activity (lead salt precipitation) and by immunohistochemistry with a polyclonal antibody raised in rabbits. Enzyme activity was found in the brush border of the proximal tubule, highest in the P1 segments with decreasing intensity in the P2 segments and weakest in P3 segments in the medullary rays of the cortex. The P3 segments of the outer stripe showed slightly higher activity. Activity was also apparent in the intercalated cells in the connecting tubule and collecting duct, whereas all other tubular and glomerular structures were negative. Activity in peritubular and perivascular connective tissue was highest in the cortical labyrinth, weak or absent in the medullary rays of the cortex, and entirely absent in the medulla. The distribution of the antigen was fully congruent with that of the enzyme activity. With respect to the role of adenosine in regulation of renal blood flow and glomerular filtration rate, the distribution of 5'-Nu in the cortical interstitium may be particularly significant. The possibility of nucleotide cleavage at the brush-border membranes may be important for salvage of nucleotides from the tubular lumen.

A monoclonal antibody IgG, has been raised against ecto-5'-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 micron thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative. The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.

5'-Nucleotidase activity was solubilized from a particulate fraction of rat renal homogenates by Sulphobetaine 14. An 11,430-fold purification was achieved by a two-step chromatographic procedure using concanavalin-A Sepharose and ADP-agarose. SDS-PAGE of the purified material revealed a single polypeptide band with a M(r) of 69,000. The enyzme exhibited absolute specificity for 5'-mononucleotides. Among 7 tested substrates, adenosine monophosphate (AMP) showed the highest value of V/K(m). The K(m) for 5'-AMP is 5.1 μmol/1 and V is 632 pmol/min/mg. The plot of activity versus pH shows a broad plateau between pH 6.8 and 8.0. The hydrolysis of 5'-AMP was competitively inhibited by adenosine 5'-triphosphate (ATP; K; = 1.2 μmol/1), adenosine 5'-diphosphate (ADP; K(i) = 0.032 μmol/1) and a,ß-methyleneadenosine 5'-diphosphate (AOPCP; K(i) = 0.005 μmol/1). All of the 5 detergents tested activated the enzyme. Sulphobetaine 14 was the most potent and resulted in a 4-fold stimulation by increasing V without change of Km. Addition of exogenous divalent cations was not required for activity. However, the enzyme was inhibited by EDTA. This inhibition was overcome by the addition of Co^2+, M^n2+ and to a lesser extent of Mg^2+. Hg^2+, Zn^2+, Cu^2+ and Pb2+ inhibited in the low micromolar range. The properties of this enzyme from the rat kidney are similar to those reported in the literature for ecto 5'-nucleotidases from other