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Elizabeth V. Menshikova - One of the best experts on this subject based on the ideXlab platform.
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characterization of the n 6 etheno bridge method to assess extracellular metabolism of adenine nucleotides detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism
Purinergic Signalling, 2020Co-Authors: Edwin K. Jackson, Zaichuan Mi, Delbert G. Gillespie, Dongmei Cheng, Elizabeth V. MenshikovaAbstract:The goal of this study was to determine the validity of using N(6)-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N(6)-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N(6)-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-Nucleotidases process N(6)-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N(6)-etheno-ATP. Applied N(6)-etheno-ATP was recovered in the medium as N(6)-etheno-ADP, N(6)-etheno-AMP, N(6)-etheno-adenosine, and surprisingly N(6)-etheno-adenine; intracellular N(6)-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N(6)-etheno-ATP, N(6)-etheno-ADP, N(6)-etheno-AMP, N(6)-etheno-adenosine, and N(6)-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors ((3)H-alpha,beta-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors ((35)S-ATP-alphaS binding to rat brain membranes), suggesting minimal pharmacological activity. N(6)-etheno-adenosine was partially converted to N(6)-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N(6)-etheno-ATP was quickly metabolized, with N(6)-etheno-adenine being the main product in naive rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N(6)-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-Nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N(6)-etheno-adenosine to N(6)-etheno-adenine.
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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
Purinergic Signalling, 2020Co-Authors: Edwin K. Jackson, Dongmei Cheng, Delbert G. Gillespie, Elizabeth V. MenshikovaAbstract:The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-Nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naive rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-Nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.
Charles R Wira - One of the best experts on this subject based on the ideXlab platform.
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effects of tenofovir on cytokines and Nucleotidases in hiv 1 target cells and the mucosal tissue environment in the female reproductive tract
Antimicrobial Agents and Chemotherapy, 2014Co-Authors: Nabanita Biswas, Marta Rodriguezgarcia, Zheng Shen, Sarah G Crist, Jack E Bodwell, John V Fahey, Charles R WiraAbstract:Tenofovir (TFV) is a reverse transcriptase inhibitor used in microbicide preexposure prophylaxis trials to prevent HIV infection. Recognizing that changes in cytokine/chemokine secretion and nucleotidase biological activity can influence female reproductive tract (FRT) immune protection against HIV infection, we tested the hypothesis that TFV regulates immune protection in the FRT. Epithelial cells, fibroblasts, CD4(+) T cells, and CD14(+) cells were isolated from the endometrium (Em), endocervix (Cx), and ectocervix (Ecx) following hysterectomy. The levels of proinflammatory cytokines (macrophage inflammatory protein 3α [MIP-3α], interleukin 8 [IL-8], and tumor necrosis factor alpha [TNF-α]), the expression levels of specific Nucleotidases, and nucleotidase biological activities were analyzed in the presence or absence of TFV. TFV influenced mRNA and/or protein cytokines and Nucleotidases in a cell- and site-specific manner. TFV significantly enhanced IL-8 and TNF-α secretion by epithelial cells from the Em and Ecx but not from the Cx. In contrast, in response to TFV, IL-8 secretion was significantly decreased in Em and Cx fibroblasts but increased with fibroblasts from the Ecx. When incubated with CD4(+) T cells from the FRT, TFV increased IL-8 (Em and Ecx) and TNF-α (Cx and Ecx) secretion levels. Moreover, when incubated with Em CD14(+) cells, TFV significantly increased MIP-3α, IL-8, and TNF-α secretion levels relative to those of the controls. In contrast, nucleotidase biological activities were significantly decreased by TFV in epithelial (Cx) and CD4(+) T cells (Em) but increased in fibroblasts (Em). Our findings indicate that TFV modulates proinflammatory cytokines, nucleotidase gene expression, and nucleotidase biological activity in epithelial cells, fibroblasts, CD4(+) T cells, and CD14(+) cells at distinct sites within the FRT.
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effect of tenofovir on Nucleotidases and cytokines in hiv 1 target cells
PLOS ONE, 2013Co-Authors: Nabanita Biswas, Marta Rodriguezgarcia, Zheng Shen, Sarah G Crist, Jack E Bodwell, John V Fahey, Charles R WiraAbstract:Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4+ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of Nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5’-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5’ nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4+ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4+ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.
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estradiol regulation of Nucleotidases in female reproductive tract epithelial cells and fibroblasts
PLOS ONE, 2013Co-Authors: Zheng Shen, Marta Rodriguezgarcia, Sarah G Crist, Jack E Bodwell, John V Fahey, Richard M Rossoll, Mickey V Patel, Charles R WiraAbstract:The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5′-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5′-Nucleotidase biological assay for Nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.
Edwin K. Jackson - One of the best experts on this subject based on the ideXlab platform.
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characterization of the n 6 etheno bridge method to assess extracellular metabolism of adenine nucleotides detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism
Purinergic Signalling, 2020Co-Authors: Edwin K. Jackson, Zaichuan Mi, Delbert G. Gillespie, Dongmei Cheng, Elizabeth V. MenshikovaAbstract:The goal of this study was to determine the validity of using N(6)-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N(6)-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N(6)-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-Nucleotidases process N(6)-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N(6)-etheno-ATP. Applied N(6)-etheno-ATP was recovered in the medium as N(6)-etheno-ADP, N(6)-etheno-AMP, N(6)-etheno-adenosine, and surprisingly N(6)-etheno-adenine; intracellular N(6)-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N(6)-etheno-ATP, N(6)-etheno-ADP, N(6)-etheno-AMP, N(6)-etheno-adenosine, and N(6)-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors ((3)H-alpha,beta-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors ((35)S-ATP-alphaS binding to rat brain membranes), suggesting minimal pharmacological activity. N(6)-etheno-adenosine was partially converted to N(6)-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N(6)-etheno-ATP was quickly metabolized, with N(6)-etheno-adenine being the main product in naive rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N(6)-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-Nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N(6)-etheno-adenosine to N(6)-etheno-adenine.
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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
Purinergic Signalling, 2020Co-Authors: Edwin K. Jackson, Dongmei Cheng, Delbert G. Gillespie, Elizabeth V. MenshikovaAbstract:The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-Nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naive rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-Nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.
Cletus J M Dsouza - One of the best experts on this subject based on the ideXlab platform.
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the pharmacological role of Nucleotidases in snake venoms
Cell Biochemistry and Function, 2010Co-Authors: B L Dhananjaya, Cletus J M DsouzaAbstract:Several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as Nucleotidases (5'nucleotidase, ATPase, and ADPase) are less studied and their pharmacological role in venoms is not clearly defined. Very few studies have shown the pharmacological importance of these endogenous purine release related enzymes in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It is suggested that their major function is in the generation of purines (mainly adenosine)-a multitoxin. Therefore, it appears that these enzymes play a central role in liberating adenosine and through the action of adenosine help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.
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snake venom nucleases Nucleotidases and phosphomonoesterases
2009Co-Authors: B L Dhananjaya, B S Vishwanath, Cletus J M DsouzaAbstract:Snake venom components, acting in concert within prey, cause immobilization and initiate diges- tion. Additional pharmacological activities have evolved among several hydrolytic enzymes of snake venom, which interfere with numerous physiological processes of the prey and which pro- duce these effects. However, hydrolytic enzymes such as nucleases (DNase, RNase, and phospho- diesterase), Nucleotidases (5 nucleotidase, ATPase, and ADPase), and phosphomonoesterases (acid and alkaline phosphomonoesterases) have not been extensively studied, and their pharmacological roles in venoms are not clearly defined. Also, they show overlapping substrate specificities and have other biochemical properties in common, producing uncertainty about their individual identity in venoms. For example, DNases, RNases, and phosphodiesterase share similar properties in substrate hydrolysis but differ in their pH optima and metal ion requirement for activity. Nucleotidases such as ATPases and ADPases have overlapping substrate specificities with phosphodiesterase. The dif- ferences among them are still not clear, and analyzing cDNA or amino acid sequences of the puri- fied enzymes is necessary to resolve these differences (if any). Except for RNases, most of these enzymes are of high molecular weight, and all except DNases and RNases are known to be metallo- enzymes. Of these, only 5 Nucleotidases and ADPases are known to be involved in inhibition of platelet aggregation and blood coagulation. However, the near-ubiquitous distribution of these enzymes in venoms suggests a significant role for these enzymes in envenomation. It is suggested that their major function may be in the generation of adenosine, a multitoxin. Adenosine generated in vivo by their synergistic action on endogenous substrates is known to bring about various phar- macological effects, similar to those induced by the whole venom. Therefore, it appears that these enzymes play a central role in liberating adenosine, and through the action of adenosine, assist in prey immobilization. In addition, these enzymes could possess other pharmacological activities, which can interfere in diverse physiological processes of the prey/victim, but this has not been veri- fied by pharmacological studies using purified enzymes. Further research is needed to characterize the biological roles of these enzymes in snake venoms and to establish clearly their role in enveno- mation sequelae
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anticoagulant effect of naja naja venom 5 nucleotidase demonstration through the use of novel specific inhibitor vanillic acid
Toxicon, 2006Co-Authors: B L Dhananjaya, B S Vishwanath, A Nataraju, R Rajesh, C Raghavendra D Gowda, B K Sharath, Cletus J M DsouzaAbstract:The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.
David P Westfall - One of the best experts on this subject based on the ideXlab platform.
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enzyme kinetics and pharmacological characterization of Nucleotidases released from the guinea pig isolated vas deferens during nerve stimulation evidence for a soluble ecto nucleoside triphosphate diphosphohydrolase like atpase and a soluble ecto 5 nucleotidase like ampase
Journal of Pharmacology and Experimental Therapeutics, 2002Co-Authors: Svetlana T Mihaylovatodorova, Latchezar D Todorov, David P WestfallAbstract:Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release not only of the cotransmitters ATP and norepinephrine but also of soluble Nucleotidases that break down extracellular ATP, ADP, and AMP into adenosine. In this study we show that the apparent K(m) values of the releasable enzyme activity vary depending on which of these adenine nucleotides is used as initial substrate. The K(m) value for ATP was 33.6 +/- 2.3 microM, 21.0 +/- 2.3 microM for ADP, and 10.0 +/- 1.1 microM for AMP. The ratios of the V(max) values for each enzyme reaction were 4:2:3. We have also found a different sensitivity of the metabolism of ATP and AMP by releasable Nucleotidases to known nucleotidase inhibitors. Suramin inhibited the breakdown of ATP by releasable Nucleotidases in a noncompetitive manner and with a K(i) value of 53 microM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor alpha,beta-methylene ADP inhibited the breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-beta,gamma-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN(3) (10 mM) affected neither reaction, but inhibited the ADP metabolism. Phosphatase- and phosphodiesterase inhibitors did not affect the activity of the releasable Nucleotidases. This evidence suggests that the soluble Nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5'-nucleotidase-like and an ecto-nucleoside triphosphate diphosphohydrolase-like activity.
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correlation between the release of the sympathetic neurotransmitter atp and soluble Nucleotidases from the guinea pig vas deferens
Journal of Pharmacology and Experimental Therapeutics, 2001Co-Authors: Svetlana T Mihaylovatodorova, Latchezar D Todorov, David P WestfallAbstract:Recently, we have shown that by releasing specific Nucleotidases the sympathetic nerves of the guinea pig vas deferens may regulate the metabolism of extracellular adenine nucleotides and consequently, the inactivation of neurotransmitter ATP. Based on the evidence for tetrodotoxin sensitivity and calcium dependence of the nerve stimulation-evoked overflow of enzyme activity, we have suggested that soluble Nucleotidases may be stored in synaptic vesicles within the sympathetic nerves and released upon arrival of nerve action potentials by a mechanism similar to that for release of neurotransmitters. To further test this hypothesis we studied the time course of nerve stimulation-evoked overflow of ATP, norepinephrine (NE), releasable ATPase (r-ATPase) activity, and releasable AMPase (r-AMPase) activity under control conditions and in the presence of drugs known to selectively modulate sympathetic neurotransmission. The results show that the time course of overflow of r-ATPase and r-AMPase activities resembles the transient pattern of overflow of ATP but not the tonic pattern of overflow of NE. Vasa deferentia dissected from animals treated with reserpine release ATP, r-ATPase, and r-AMPase, whereas the overflow of NE is completely abolished. Guanethidine, on the other hand, inhibits equally well the overflow of the two neurotransmitters and the releasable nucleotidase activities. Agonists of the α2-adrenergic receptors abolish the overflow of ATP, r- ATPase, and r-AMPase but not the overflow of NE. This evidence supports the idea that the sympathetic nerves of the guinea pig vas deferens store and release ATP together with specific Nucleotidases responsible for the inactivation of this neurotransmitter.
