A commonly used ecto-ATPase inhibitor, ARL-67156, blocks degradation of ADP more than the degradation of ATP in murine colon

Abstract

Background: Adenosine 5'-triphosphate (ATP) is released extracellularly as a neurotransmitter and an autocrine or paracrine mediator in numerous systems, including the gastrointestinal tract. It is rapidly degraded to active and inactive metabolites by membrane-bound enzymes. Investigators frequently use inhibitors of ATP hydrolysis such as ARL-67156 and POM-1 to suppress the catabolism of ATP and prolong its effects in pharmacological studies. Our aim was to investigate directly the effects of ARL-67156 and POM-1 on the degradation of ATP and adenosine 5'-diphosphate (ADP) in mouse colonic muscles.

Methods: The degradation of ATP and ADP was evaluated by superfusing tissues with 1,N(6) -etheno-ATP (eATP) and 1,N(6) -etheno-ADP (eADP) as substrates and monitoring the decrease in substrate and increase in products (i.e., eADP, eAMP, and e-adenosine) by high-performance liquid chromatography techniques with fluorescence detection. Relaxation responses to etheno-derivatized and non-derivatized ATP and ADP were examined in isometric tension experiments.

Key results: ARL-67156 inhibits the degradation of ADP but not of ATP, whereas POM-1 inhibits the degradation of ATP but not of ADP in murine colonic muscles. Consequently, ARL-67156 enhances relaxation responses to both ATP and ADP, whereas POM-1 reduces relaxation to ATP and does not affect relaxation to ADP.

Conclusions & inferences: Studies that use ARL-67156 to inhibit ATP degradation in smooth muscle likely evaluate responses to accumulated ADP rather than ATP. POM-1 appears to be a more selective inhibitor of ATP degradation in the mouse colon. The choice of pharmacological tools in studies on extracellular ATP signaling may affect the interpretation of experimental data in functional studies.

Keywords: ADP; ARL-67156; ATP; ATP degradation; POM-1; colon; nucleotidases.

Figure 1. Metabolism of non-derivatized and 1,N⁶-etheno-derivatized ATP and enzyme kinetics in murine colon

(A) Chemical structures of non-derivatized ATP (top) and 1,N⁶-etheno-derivatized ATP (eATP, bottom). Cleavage sites for NTPDases and 5’nucleotidase, indicated by the scissors signs, remain unchanged by etheno-derivatization. (B) Superfusion of murine colon tissues with 50 nM eATP substrate (upper chromatogram) results in formation of metabolites eADP, eAMP and e-adenosine, eADO. Likewise, superfusion of tissues with 50 nM non-derivatized ATP substrate (bottom chromatogram), followed by etheno-derivatization after the enzymatic reaction, results in production of eADP, eAMP and eADO. LU, luminescence units. (C) Diagram of reaction rate and Michaelis-Menten kinetics of ATPase activities in the murine colon assessed by the decrease in eATP substrate (10-200 nM) after contact with tissue for 30 s; n=6-12. (D) Diagram of reaction rate and Michaelis-Menten kinetics of ADPase activities in the murine colon assessed by the decrease in eADP substrate (10-200 nM) after contact with tissue for 30 s; eADP n=2-9. In both (C) and (D) the velocity of substrate reduction (pmol.mg tissue⁻¹.min⁻¹) was plotted against substrate concentration [nM]. The K_m values (in nM) are indicated on the graphs.

Figure 2. Degradation of eATP in the murine colon

(A) Original chromatograms of eATP (50 nM) in the absence of tissue [(−) tissue] (blue trace), in presence of tissue with no drug [(+) tissue] (red trace), and in the presence of tissue pre-treated with either ARL-67156 (100 μM) (green trace) or POM-1 (100 μM) (purple trace). Note that the eATP substrate contained small amount of eADP, negligible amount of eAMP and no eADO. eATP was decreased and eADP, eAMP and eADO were increased in the (+) tissue samples. In the presence of ARL-67156, the decrease in eATP was unchanged, eADP was increased more than in (+) tissue control, and the formation of eAMP and eADO were decreased. In the presence of POM-1, the decrease of eATP and the formation of eADP, eAMP and eADO were inhibited. LU, luminescence units (B) Graphic representation of substrate (eATP 50 nM) reduction and product (eADP+eAMP+eADO) formation in (−) tissue control, (+) tissue control, (+) tissue plus ARL-67156 (100 μM) and (+) tissue plus POM-1 (100 μM). Asterisks denote significant differences from the amounts of substrate or products in (−) tissue samples (***P<0.001); number of experiments in parenthesis. (C) Graphic representation of product (eADP+eAMP+eADO) formation from 25 nM eATP substrate in (−) tissue control, (+) tissue control, (+) tissue plus ARL-67156 (100 μM) and (+) tissue plus POM-1 (100 μM). Asterisks denote significant differences from the amounts of products in (−) tissue samples (***P<0.001); open circle denotes significant difference from the amounts of e-products in (+) tissue control samples (^oP<0.05); number of experiments in parentheses.

Figure 3. Degradation of eADP in murine colon

(A) Original chromatogram of eADP (50 nM) in the absence [(−) tissue] (blue trace), in presence of tissue [(+) tissue, 30 s contact of substrate with tissue] (red trace) and in tissue being in contact with either ARL-67156 (100 μM) (green trace) or POM-1 (100 μM) (purple trace). Note that the eADP substrate contained negligible amounts of eAMP and no eADO. eADP was decreased and eAMP and eADO were increased in the (+) tissue samples (red trace). In the presence of ARL-67156, the decrease in eADP and formation of eAMP and eADO were inhibited. In the presence of POM-1, the decrease of eADP and formation of eAMP and eADO were unchanged compared with (+) tissue control. LU, luminescence units (B) Graphic representation of substrate (eADP 50 nM) reduction and product (eAMP+eADO) formation in (−) tissue control, (+) tissue control, (+) tissue plus ARL-67156 and (+) tissue plus POM-1. Asterisks denote significant differences from the amounts of substrate or e-products in (−) tissue controls (***P<0.001, **P<0.01); number of experiments in parenthesis. (C) Graphic representation of product (eAMP+eADO) formation from 25 nM eADP substrate in (−) tissue control, (+) tissue control, (+) tissue plus ARL-67156 (100 μM) and (+) tissue plus POM-1 (100 μM). Asterisks denote significant differences from the amounts of e-products in (−) tissue samples (***P<0.001, **P<0.01); number of experiments in parentheses.

Figure 4. ARL-67156 potentiates the relaxation responses to ATP and ADP whereas POM-1 reduces the response to ATP and has no effect on response to ADP

(A) Original traces of spontaneous contractions in mouse isolated colonic muscle. Black lines under the traces indicate when ATP or ADP was added to the bath. Inhibitors were added to the bath for 25 mins before ATP or ADP were added (presence of inhibitors indicated by black lines above traces); w, washout. (B) Summaries of contractile responses to ATP or ADP in the absence and presence of ARL-67156 (100 μM) and POM-1 (100 μM) as area under the trace (AUT). Asterisks denote significant differences from the responses in the absence of inhibitors (**P<0.01, ***P<0.001); number of experiments in parentheses.

Figure 5. ARL-67156 potentiates the relaxation responses to eATP and eADP whereas POM-1 reduces the response to eATP and has no effect on the response to eADP

(A) Original traces of spontaneous contractions in mouse isolated colonic muscle. Black lines under the traces indicate when eATP or eADP were added to the bath. Inhibitors were added to the bath for 25 mins before eATP or eADP were added (presence of inhibitors indicated by black lines above traces); w, washout. (B) Summaries of contractile responses to eATP or eADP in the absence and presence of ARL-67156 (100 μM) and POM-1 (100 μM) as area under the trace (AUT). Asterisks denote significant differences from the responses in the absence of inhibitors (***P<0.001); number of experiments in parentheses.