Regional Differences in Extracellular Purine Degradation in the Prostatic and Epididymal Portions of the Rat Vas Deferens

Abstract

1. The aim of the present study was to compare ecto-nucleotidase activities in rat bisected vas deferens using 1,N6-etheno(epsilon)-nucleotides (epsilon-ATP and epsilon-AMP) as substrates. Degradation was estimated by measuring the disappearance of the substrate and the appearance of its metabolites using HPLC with fluorescence detection. Incubation of tissue preparations (prostatic or epididymal portions) with 300 nmol/L epsilon-ATP at 37 degrees C caused a partial disappearance of epsilon-ATP and appearance of its metabolites (epsilon-ADP, epsilon-AMP and epsilon-adenosine). Incubation at 25 degrees C reduced epsilon-ATP degradation more in the prostatic than in the epididymal portion. 2. Incubation of tissue preparations with epsilon-AMP at 37 degrees C resulted in the disappearance of epsilon-AMP and the appearance of epsilon-adenosine, which was more pronounced in the epididymal than in the prostatic portion. Incubation at 25 degrees C reduced epsilon-AMP degradation more in the epididymal than in the prostatic portion. 3. Decreasing pH from 7.4 to 6.5 enhanced epsilon-AMP degradation only in the prostatic portion, whereas increasing pH from 7.4 to 8.5 enhanced epsilon-AMP degradation in both portions, but more markedly in the epididymal portion. The alkaline phosphatase inhibitors levamisole (10 mmol/L) and beta-glycerophosphate (10 mmol/L) reduced epsilon-AMP degradation only in the epididymal portion. 4. In conclusion, the results of the present study are compatible with the presence, in the bisected rat vas deferens, of an ecto-nucleotidase system that is involved in the degradation of extracellular purines, which may differ between the epididymal and prostatic portions, with the epididymal portion presenting a different and higher capacity to form adenosine.

Lack of Endogenous Adenosine Tonus on Sympathetic Neurotransmission in Spontaneously Hypertensive Rat Mesenteric Artery

“…The following drugs were used: levo-[ring-2,5,6-3H]-noradrenaline, specific activity 41.3 Ci/mmol, was from DuPont NEN (I.L.C., Lisboa, Portugal); desipramine hydrochloride, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261), S-(4-Nitrobenzyl)-6-thioinosine (NBTI) and 5-Iodotubericidin (ITU), (8R)-3-(2-Deoxy-β-D-erythro-pentofuranosyl)-3,4,7,8-tetrahydroimidazo[4,5-d][1], [3]diazepin-8-ol (pentostatin), α,β-methylene ADP, N6-cyclopentyladenosine (CPA), 2-p–(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) were purchased from Sigma-Aldrich (Sintra, Portugal). The following antibodies were used: rabbit polyclonal anti-A1 (epitope corresponding to amino acids 287-326 mapping at the C-terminus of human adenosine A 1 receptors; sc-28995), anti-A 2A (epitope corresponding to amino acids 331-412 mapping at the C-terminus of human adenosine A 2A receptors; sc-13937) were purchased from Santa Cruz Biotechnology, Inc., CA, USA; mouse monoclonal anti-tyrosine hydroxilase antibody (TH(45): sc-136100, Santa Cruz Biotechnology, Inc., CA, USA and MAB318, Millipore Corporation, CA, USA); anti-glial fribillary acidic protein (GFAP) mouse monoclonal antibody (G6171, Sigma-Aldrich, Inc., USA) and rabbit GFAP polyclonal antibody (18-0063, Invitrogen, Life Technologies, SA, Madrid, Spain).…”

“…[3], [16]–[19] Briefly, mesenteric artery segments were superfused (Krebs-Henseleit, 1 mL.min −1 ) and electrically stimulated twice (S 1 -S 2 ; 5 Hz, 100 pulses, 1 ms, 50 mA) 30-min apart ( t = 90 min and t = 120 min). 5-min superfusates were collected and heated at 80°C.…”

“…ATP is then sequentially dephosphorylated into ADP, AMP and adenosine. [3] Besides its action at the synapse, adenosine may function as a non-synaptic signalling molecule upon diffusion from its local of origin influencing neurotransmission, inflammation and immune responses [4]. Adenosine effects occur through activation of four G-protein coupled receptors, adenosine A 1 , A 2A , A 2B and A 3 receptors [5]…”

Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

“…It is not surprising, therefore, that a number of studies used etheno-bridged adenine nucleotides to evaluate the role of ecto-nucleotidases in the metabolism of extracellular adenine nucleotides. For example, this method has been employed to study ecto-nucleotidases in the guinea-pig vas deferens [52], intestinal epithelial cells [53], human microvascular endothelial cells [54], canine mesenteric artery and vein [55], the Langendorff heart preparation [56], the rat vas deferens [57], regulatory T cells [58], melanoma cells [59], macrophages [60], and epicardium-derived cells [61].…”