Ecto-ATPases (E C 3.6.1.15.)

Ecto-ATPase, or E-type ATPase catabolises nucleotide triphosphates with broad specificity for adenine and uridine nucleotides and are distinct from the intracellular ion-pump ATPases and the mitochondrial ATPases which selectively dephosphorylate ATP as their substrate. In addition the classical inhibitors o f intracellular ATPases, vanadium ions, ouabain or oligomycin have no effect on the activity o f extracellular ATPases. It is believed that this is the major enzyme responsible for degradation of ATP in the bladder and hence inactivation of the contractile effects o f this neurotransmitter (Hourani and Chown, 1988 ; Ziganshin et a/., 1994).

The ecto-ATPase protein has proved difficult to purify and characterise, partly due to the low abundance of the protein on the cell surface and partly due to its susceptibility to inactivation by many detergents commonly used in enzyme purification methods (Plesner, 1995). In spite o f these difficulties. E-type ATPases have been purified from numerous tissues including rat liver (Lin, 1985; Lin, 1990), rabbit skeletal muscle (Treuheit et al., 1992), chicken gizzard (Stout et al., 1995) and activated NK cells (Dombrowski et al., 1993). Rather than ecto-ATPase activity being due to a single enzyme, it is much more probable that a family of related proteins exists. For example, the rat liver plasma membrane ecto-ATPase is a 57kDa glycoprotein consisting of 519 amino acids (Lin & Guidotti, 1989), the rabbit skeletal muscle and chicken gizzard enzymes are monomers of 6 6kDa.

Dombrowski et al. (1997) demonstrated that ecto-ATPase is a marker o f B cell activation expressed by immortalised human and murine B cells. Characterisation o f the kinetic parameters of the B cell ecto-ATPases and immunological crossreactivity of antibodies generated against the chicken gizzard ecto-ATPase demonstrated that there was significant interspecies conservation o f the enzymic and immunological molecular characteristics in this class of enzymes (Stout et al., 1995).

Ecto-ATPase activity has also been demonstrated on visceral smooth muscle cells. The structure-activity relationships for these enzymes have been determined in the guinea- pig taenia caeci and urinary bladder (Welford et al, 1986; 1987) and ATP, ADP and AMP are degraded to adenosine. Purine ring-substituted analogues, such as 2- methylthioadenosine triphosphate (2-MeSATP) had a similar rate o f dephosphorylation to ATP. Stereoselectivity was observed for the D- over the L-enantiomer, in a manner similar to that of pig aortic endothelial cells (Pearson et al, 1980; 1985). In addition, the purine nucleoside triphosphates ATP and guanosine 5’-triphosphate (GTP), and the pyrimidine nucleoside triphosphates uridine 5’-triphosphate (UTP) and cytidine 5’- triphosphate (CTP) were all degraded at a similar rate to their corresponding nucleosides. Non-terminally substituted phosphorothioate analogues, such as ATP-p-S and ADP-a-S also showed stereoselectivity between the two diastereoisomeric forms, whereas the terminally substituted analogues ATP-y-S and ADP-p-S, were resistant to degradation (Welford gr a/., 1986; 1987).

In addition, the methylene-substituted derivatives of ATP and ADP, a,P-me-ATP, p,y- me-L-ATP and adenosine 5’-(a, p-methylene)diphosphonate (AMPCP), were found to

be completely resistant to degradation from the point at which the methylene group replaces the oxygen in the phosphate chain (Welford et al, 1986; 1987). However, the guinea-pig taenia caeci was unable to dephosphorylate adenosine 5’-0-(l- thiotriphosphate (ATP-a-S) (Welford et al, 1986) whereas the guinea-pig urinary bladder could degrade this analogue (Welford et al, 1987), although at a much slower rate than for ATP.

Smooth muscle cells and endothelial cells have been shown to dephosphorylate ATP via ecto-ATPase with similar activity, although in smooth muscle cells the production of adenosine occurs rapidly, whereas in endothelial cells adenosine is produced much more slowly. (Gordon et al, 1986; 1989).

Ecto-ATPase activity in primary cultures of guinea-pig vas deferens smooth muscle cells has been demonstrated by measurement of the production o f inorganic phosphate

(Pi) from ATP. The ecto-ATPase activity was found to be insensitive to a number o f inhibitors, including ouabain, oligomycin, sodium azide, p-nitrophenol and (3- glycerophosphate. The enzyme activity was found to be Ca^^- and Mg^"^ -dependent. The same study revealed that antagonists o f P2X receptors suramin, pyridoxal- phosphate-6-azophenyl-2,4’-disulphonic acid (PPADS), 4,4’-diisothiocyanostilbene- 2,2’-disulphonic acid (DIDS) and pyridoxal-5-phosphate significantly inhibited ecto- ATPase activity, whereas the PI receptor antagonists DPCPX and 8-SPT had no effects

on the activity of the enzyme (Ziganshin et al, 1995).

a number o f foreign membrane receptors and ion channels (Snutch et a l, 1988). In addition, Xenopus oocytes possess native receptors for ATP on their enveloping follicle cell layer (Lotan et al, 1986). Ziganshin et a l (1994a) demonstrated the presence of an oocyte ecto-ATPase which limits the potency of agonists at the native receptor and set about characterising the properties of the enzyme activity. They described an ecto- enzyme with a broad substrate specificity for ATP, ADP and AMP, which was highly dependent on extracellular Ca^^ and Mg^^. Further, the ecto-ATPase activity o f the oocyte was inhibited by the P2 receptor antagonists suramin, PIT and TNP-ATP (Ziganshin et al, 1995). The inhibitory action on extracellular ATP degradation of some P2 receptor antagonists, such as suramin, DIDS and Reactive blue 2 (or Cibacron blue), had previously been reported in smooth muscle cells, endothelial cells and hepatocytes (Knowles, 1988; Hourani & Chown, 1989; Yagi et a l, 1994). The PI receptor antagonist 8-SPT had no effect on the rate o f degradation o f either ATP or ADP.

A recently identified selective inhibitor o f ecto-ATPase, FPL 67156 (6-N,N-diethyl-D-

P,y-dibromo-methyleneATP) now known as ARL 67156 is a structural analogue o f ATP (Beukers et a l, 1994). Studies have shown that in some tissues ARL 67156 caused a leftward shift of the concentration effect curves to the hydrolysable agonists ATP and UTP (Crack et al, 1995). In the rabbit ear artery the potency order for agonists at P2X receptors was p,y-me-ATP » ATPyS > 2MeSATP> ATP> UTP, whereas in the presence o f ARL 67156 the potency order for these same agonists was altered to: p,y- me-ATP > 2MeSATP = ATP> ATPyS > UTP. In the same study it was found that the potency order for agonists at P2Y receptors on guinea-pig aortic rings was: 2MeSATP

» ATPyS > ATP » AMPCPP = UTP. However, in the case o f P2Y receptors the potency order of agonists were unchanged in the presence o f ARL 67156 (Crack er al,

1995). In both the rabbit ear artery and the guinea-pig aorta leftward shifts in the agonist concentration-effect curves together with the alterations in the agonist potency orders at P2X receptors may indicate that there are different levels o f ecto-ATPase activity in the two tissues.

The P2 receptor agonists ATP-y-S, a,p-M eATP and AMPPNP have been reported to inhibit the ecto-ATPase activity of bovine artery endothelial cells. The p/Cjo values for these ligands were found to be 5.2, 4.5 and 4.0 respectively (Chen & Lin, 1997). In addition, the selective ecto-ATPase inhibitor ARL 67156 inhibited ecto-ATPase activity in these cells with a p/Cjo value of 4.0. Each o f the above agonists also exhibited high agonist potency for causing increased phosphoinositide (PI) turnover in these cells and the authors concluded from this that their action as ecto-ATPase inhibitors accounted for their high agonist potency.

The release o f specific ectonucleotidases represents an as yet novel mechanism for the termination of neurotransmitter action (Todorov et al, 1997). On superfusion of the guinea-pig vas deferens with exogenous ATP almost no detectable degradation occurred. However, on stimulation o f the sympathetic nerves the exogenous ATP was almost all degraded. Furthermore, when neurotransmission was inhibited by the addition o f Cd^^ or omission of Ca^^ no appreciable breakdown was observed. In addition, superfusate collected during nerve stimulation was able to degrade ATP when added latterly. This effect was lost on addition o f tetrodotoxin, guanethidine or omission

of from the perfusate during nerve stimulation. Further evidence that nerve stimulation was the mechanism by which ectonucleotidases were being released from the nerves was that exogenously added ATP, AMPCPP, noradrenaline, methoxamine and phenylephrine all caused the guinea-pig vas deferens to contract but failed to produce release of soluble ectonucleotidases into the bathing medium. Finally, soluble ectonucleotidase activity was not inhibited by known inhibitors o f intracellular ATPases, such as vanadium, oligomycin or sodium azide, whereas ARL 67156 (lOOpM) reduced the soluble ATPase activity by approximately 50% (Todorov et a l, 1997).

Soluble ectonucleotidases are believed to be stored within vesicles in the nerve endings, since their release is Ca^^-dependent and TTX sensitive. It is assumed that ATP and nucleotidase enzymes are not co-stored since this would be likely to result in vesicular breakdown o f ATP and that a heterogeneous population of synaptic vesicles exist in sympathetic nerves (Todorov et a l, 1996). However, it has been suggested that the ATP and nucleotidases are co-stored, but that the enzymes are in an inactive state due to the acidic pH environment within the vesicles (Schuldiner et a l, 1995). On release into the less acidic extracellular compartment the soluble ectonucleotidases become activated. Electrical stimulation of the guinea-pig vas deferens (Todorov a l, 1997), guinea-pig urinary bladder, rat vas deferens, mouse vas deferens and rabbit vas deferens (Westfall

et al, 2000a; b), but not the guinea-pig taenia coli (Westfall et a l, 2000a) results in the release o f ATP and NA as cotransmitters and also ectonucleotidase enzymes which degrade ATP to adenosine, with the greatest enzyme activity observed on stimulation o f the guinea-pig vas deferens. It is not clear whether these differences in activity reflect differences in the density of innervation o f these tissues or if other factors may be

involved. The molecular identity of this ecto-ATPase activity is yet to be elucidated, although it has similar characteristics to ectonucleoside triphosphate diphosphohydrolases (Westfall e ta l, 2000a).

The vertebrate ectoATPases, present in muscular tissues and reported in rat skeletal muscle (Beeler et al 1983), rabbit skeletal muscle (Hidalgo, et al, 1983), chicken gizzard smooth muscle (Stout and Kirley, 1994) and guinea pig bladder (Hourani and Chown, 1988 ; Ziganshin et a/,,1994) have now been renamed eNTPDase2 in a recent work by Zimmermann et al, 1999. In this nomenclature eNTPDase2 is a member o f the eNTPDase (ecto-nucleoside triphosphate diphosphohydrolase) family which includes not only the integral membrane ecto-ATPases but also ectoATPDases [ecto-apyrases] and the soluble, excreted exo-ATPases. The rabbit enzyme was the first vertebrate eNTDPase purified to homogeneity (Treuheit et a l 1992) and exhibits Michaelis- Menton enzyme kinetics and is stable, like the other subfamilies o f the E-type ATPases (eNTPDases) including eNTPDase 1 (CD39) and eNTPDase3 (also known as CD39L3 and HB6). The chicken, rat and human ectoATPases (eNTPDase2), do not, however

follow these kinetics and show time dependent inactivation in the presence of ATP substrate (Hicks-Berger and Kirley, 2000). This inactivation was blocked by the lectin concanavalin A and the chemical cross-linker disuccinimidyl suberate, agents that stabilise monomer-monomer interactions, suggesting that the native state is oligomeric, presumably tetrameric (Hicks-Berger and Kirley, 2000). These authors also showed that the human ecto-ATPase depends on glycosylation for its activity and concluded that glycosylation is needed for formation and maintenance o f the correct tertiary and quaternary structure o f the enzyme. In this study chimeras o f the protein with human

CD39 (eNTDPase 1) were constructed and the two chimeras produced demonstrated that it is the N-terminal half o f the protein that determines the relative activities o f the nucleoside di- and triphosphatases.

1.9.2, Ecto-ADPases (EC 3,6. L 6,)

The following nucleotidases (sections 1.9.2-1.9.4) have not been well characterised in the bladder and are included solely to complete this description of the metabolism of ATP to adenosine.

Ecto-ADPases have been characterised in vascular endothelial cells and have been shown to be inhibited by nucleotides such as AMPPCP and ATP-y-S (Pearson et a l,

1986). In these cells their physiological role is to counteract the thrombotic response to vascular injury by metabolising the platelet released agonist ADP and thereby oppose platelet activation (Marcus et al, 1991).