Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells throug h two subtypes of purinoreceptors, P-2Y2 and P-A1. The signals to purinorec eptors are usually terminated by the action of ectonucleotidases. To demons trate these enzymatic activities, we cultured rat Sertoli cells for four da ys and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenos ine 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 marke dly 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 th at acts on the degradation of tri- and diphosphate nucleosides on the surfa ce 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 AD P, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine d eaminase 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 f or the physiological control of extracellular levels of nucleotides and nuc leosides inside the seminiferous tubules.