Ectonucleotidase activities in Sertoli cells from immature rats

Abstract

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P 2Y2 and P A1 . The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri-and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 ± 6 and 21 ± 2 nmol Pi mg -1 min -1 for ATP and ADP, respectively). The ecto-5-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 ± 2 nmol Pi mg -1 min -1 for AMP and 1.52 ± 0.13 nmol adenosine mg -1 min -1 , respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.

Genomic-independent action of thyroid hormones on NTPDase activities in Sertoli cell cultures from congenital hypothyroid rats

“…Sertoli cells were isolated from the testis of 18-day-old Wistar rats by sequential enzymatic digestion, as described by Casali et al (2001). The testes were sequentially digested with 0.25% trypsin and DNase (10 μg/ml) for 30 min at 37°C to remove the interstitial tissue.…”

“…The Sertoli cell monolayers were incubated as previously described by Casali et al (2001). Briefly, the Sertoli cell monolayers were washed 3 times with the reaction medium containing 135 mM NaCl, 5 mM KCl, 10 mM glucose and 10 mM HEPES, 2 mM CaCl 2 pH 7.4.…”

“…All assays were performed in quadruplicate. All enzyme conditions of this assay were previously described (Casali et al, 2001) in order to ensure the linearity of reaction.…”

“…Ecto-nucleotidase activities, previously described in Sertoli cell cultures, are able to promote the hydrolysis of the extracellular nucleotides ATP, ADP and AMP to adenosine (Casali et al, 2001). These enzymatic activities can regulate the extracellular concentration of adenine nucleotides and nucleosides, modulating their local effects (Filippini et al, 1994;Casali et al, 2001;Ko et al, 2003).…”

“…These enzymatic activities can regulate the extracellular concentration of adenine nucleotides and nucleosides, modulating their local effects (Filippini et al, 1994;Casali et al, 2001;Ko et al, 2003).…”