Hydrolysis of Extracellular Adenine Nucleotides by Equine Epidydimal Spermatozoa

Minelli, Alba; Moroni, Monica; Trinari, D.; Mezzasoma, I

doi:10.1016/s0305-0491(97)00213-7

Cited by 7 publications

(6 citation statements)

References 17 publications

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“…The modulation of the levels of different nucleotides may involve cross-talking between the diverse pathways activated in purinergic receptor subtypes. The general way to control the concentration of adenine nucleotides is the sequential activity of ecto-ATPases, ecto-ATP diphosphohydrolases (apyrase) and ecto-5-nucleotidases (12)(13)(14)(15)(16)(17)19,20,30). The adenosine released from cells or resulting from extracellular ATP hydrolysis can be deaminated by the ectoadenosine deaminase activity, producing inosine (18,34,36).…”

Section: The Physiological Rolementioning

confidence: 99%

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Ectonucleotidase activities in Sertoli cells from immature rats

Casali

Silva

Gelain

et al. 2001

Braz J Med Biol Res

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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.

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Section: The Physiological Rolementioning

confidence: 99%

“…The extracellular hydrolysis of ATP to adenosine by ectonucleotidases has been reported for several cell types (12)(13)(14)(15)(16)(17)(18)(19)(20). These enzymatic activities can regulate the extracellular concentration of adenine nucleotides and nucleosides modulating their local effects.…”

Section: Introductionmentioning

confidence: 99%

Ectonucleotidase activities in Sertoli cells from immature rats

Casali

Silva

Gelain

et al. 2001

Braz J Med Biol Res

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“…Thus, the Na ϩ /H ϩ exchanger should be a major candidate involved in Na ϩ and fluid reabsorption by the epididymis. It is conceivable that a high concentration of nucleosides exists in the epididymal fluid, and they could be derived from the hydrolysis of nucleotides by 5Ј-ectonucleotidase, which has been found on the plasma membranes of spermatozoa [23][24][25] and epididymal epithelial cells [26]. This study shows that the Na ϩ /nucleoside cotransporter may also contribute to Na ϩ and fluid reabsorption across the epididymal tubules.…”

Section: Discussionmentioning

confidence: 66%

Na+ Reabsorption in Cultured Rat Epididymal Epithelium via the Na+/Nucleoside Cotransporter1

Leung

Cheung

Tse

et al. 2001

Biology of Reproduction

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The effect of nucleoside on Na+ reabsorption via Na+/nucleoside cotransporter in cultured rat epididymal epithelia was studied by short-circuit current (Isc) technique. Guanosine added apically stimulated Isc in a dose-dependent manner, with a median effective concentration (EC50) of 7 +/- 2 microM (mean +/- SEM). Removal of Na+ from the apical bathing solution or pretreatment with a nonspecific Na+/nucleoside cotransporter inhibitor, phloridzin, completely blocked the Isc response to guanosine. Moreover, the guanosine response was abolished by pretreatment of the tissue with ouabain, a Na+/K+-ATPase inhibitor, suggesting the involvement of Na+/nucleoside cotransporter on the apical side and Na+/K+-ATPase on the basolateral side in Na+ reabsorption. In contrast, the Isc response to guanosine was not affected after desensitization of purinoceptors by ATP. Addition of the Na+/K+/2Cl- symport inhibitor bumetanide to the basolateral side or the nonspecific Cl- channel blocker diphenylamine-2-carboxylate to the apical side showed no effect on the Isc response to guanosine, excluding stimulation of Cl- secretion by guanosine as the cause of the guanosine-induced Isc. The Isc response to purine nucleoside (guanosine and inosine) was much higher than that to pyrimidine nucleoside (thymidine and cytidine). Consistent with substrate specificity, results of reverse transcription-polymerase chain reaction revealed mRNA for concentrative nucleoside transporter (CNT2), which is a purine nucleoside-selective Na+/nucleoside cotransporter in the epididymis, but not for CNT1. It is suggested that the Na+/nucleoside cotransporter (i.e., CNT2) may be one of the elements involved in Na+ and fluid reabsorption in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.

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“…The resulting nucleotides are further degraded by ecto-nucleotidases also present on the surface of these membrane vesicles [32,34]. The last extracellular product, adenosine, is then ef®ciently transported into sperm cells [36] or interacts with adenosine receptors present at the surface of these cells [38±41].…”

Section: Discussionmentioning

confidence: 99%

“…Determinations of diadenosine polyphosphates and their hydrolysis products were carried out with paired-ion chromatography using as mobile phase 10 mM KH 2 PO 4 , 2 mM tetrabutylammonium, ®nal pH 7.5 and 12% acetonitrile [24,25]. Adenosine was determined with reversephase chromatography with 0.1 M KH 2 PO 4 pH 6.0 and 10% methanol [36]. Data were analyzed by use of TurboChrom software (Perkin Elmer).…”

Section: Methodsmentioning

confidence: 99%