Dynamic recycling of extracellular ATP in human epithelial intestinal cells

Intestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes and food digestion. The function of these cells is strongly influenced by purinergic signalling activated by extracellular ATP (eATP) and other nucleotides. The activity of several ecto-enzymes determines the dynamic regulation of eATP. In pathological contexts, eATP may act as a danger signal controlling a variety of purinergic responses aimed at defending the organism from pathogens present in the intestinal lumen. In this study, we characterized the dynamics of eATP on polarized and non-polarized Caco-2 cells. eATP was quantified by luminometry using the luciferin-luciferase reaction. Results show that non-polarized Caco-2 cells triggered a strong but transient release of intracellular ATP after hypotonic stimuli, leading to low micromolar eATP accumulation. Subsequent eATP hydrolysis mainly determined eATP decay, though this effect could be counterbalanced by eATP synthesis by ecto-kinases kinetically characterized in this study. In polarized Caco-2 cells, eATP showed a faster turnover at the apical vs the basolateral side. To quantify the extent to which different processes contribute to eATP regulation, we created a data-driven mathematical model of the metabolism of extracellular nucleotides. Model simulations showed that eATP recycling by ecto-AK is more efficient a low micromolar eADP concentrations and is favored by the low eADPase activity of Caco-2 cells. Simulations also indicated that a transient eATP increase could be observed upon the addition of non-adenine nucleotides due the high ecto-NDPK activity in these cells. Model parameters showed that ecto-kinases are asymmetrically distributed upon polarization, with the apical side having activity levels generally greater in comparison with the basolateral side or the non-polarized cells. Finally, experiments using human intestinal epithelial cells confirmed the presence of functional ecto-kinases promoting eATP synthesis. The adaptive value of eATP regulation and purinergic signalling in the intestine is discussed.

Intestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes and food digestion. The function of these cells is strongly influenced by purinergic signalling activated by extracellular ATP (eATP) and other nucleotides. The activity of several ecto-enzymes determines the dynamic regulation of eATP. In pathological contexts, eATP may act as a danger signal controlling a variety of purinergic responses aimed at defending the organism from pathogens present in the intestinal lumen.
In this study, we characterized the dynamics of eATP on polarized and non-polarized Caco-2 cells. eATP was quantified by luminometry using the luciferin-luciferase reaction. Results show that non-polarized Caco-2 cells triggered a strong but transient release of intracellular ATP after hypotonic stimuli, leading to low micromolar eATP accumulation. Subsequent eATP hydrolysis mainly determined eATP decay, though this effect could be counterbalanced by eATP synthesis by ecto-kinases kinetically characterized in this study. In polarized Caco-2 cells, eATP showed a faster turnover at the apical vs the basolateral side.
To quantify the extent to which different processes contribute to eATP regulation, we created a data-driven mathematical model of the metabolism of extracellular nucleotides. Model simulations showed that eATP recycling by ecto-AK is more efficient a low micromolar eADP concentrations and is favored by the low eADPase activity of Caco-2 cells. Simulations also indicated that a transient eATP increase could be observed upon the addition of non-adenine nucleotides due the high ecto-NDPK activity in these cells. Model parameters showed that ecto-kinases are asymmetrically distributed upon polarization, with the apical side having activity levels generally greater in comparison with the basolateral side or the non-polarized cells.

Introduction
The surface of the intestine is covered by a layer of cells that form the intestinal epithelium.
Intestinal epithelial cells play important roles in the absorption of nutrients, secretion of electrolytes, digestion of food and host defence mechanisms [1,2]. The function of intestinal epithelial cells is strongly influenced by extracellular nucleotides, supporting a complex signalling network that mediates short-term functions such as secretion and motility, and long-term functions like proliferation and apoptosis [3,4]. Among these nucleotides, extracellular ATP (eATP) was found to be an early danger signal response to infection with enteric pathogens that eventually promote inflammation of the gut [4,5]. An important source of eATP is the intracellular ATP (iATP) found in the cytosol and vesicles of many cell types [6]. Activation of iATP release was found in subepithelial intestinal fibroblasts, human epithelial cell lines and enteroendocrine cells in response to several stimuli, including agents that elevate cAMP, such as forskolin and cholera toxin [7], low medium phosphate, hypoosmotic swelling and bacterial infection [7,8]. Currently, several mechanisms have been postulated to mediate regulated iATP release, and these mechanisms can vary according to the cell type and the stimuli [6]. For example, after a hypotonic shock, Schwann cells release ATP via the anionic channel pannexin-1 [9], while the treatment with lipopolysaccharide (LPS) induces ATP release via connexin-43 in macrophages [10]. Additionally, the vesicular release pathway for ATP was also described, as in the case of endothelial cells under hypoxia [11].
Extracellular ATP and other di-and tri-phosphonucleosides can activate purinergic receptors 2 (P2 receptors) unevenly distributed in the small and large intestine [12]. Purinergic signalling is controlled by membrane bound ecto-nucleotidases and ecto-kinases capable of promoting the synthesis and/or hydrolysis of eATP, and/or its conversion into other extracellular nucleotides and nucleosides. For any cell type and metabolic context, a specific set of ecto-enzymes may control the rate, amount and timing of nucleotide turnover [13].
Ecto-nucleoside triphosphate diphosphohydrolases (Ecto-NTPDases) are a family of enzymes promoting the extracellular hydrolysis of eATP, eADP, eUTP and eUDP. One or more members of this family are present in almost every cell. Ecto-NTPDase-1, -2, and -3, which differ regarding the specific preferences for nucleotides, are responsible for the hydrolysis of nucleoside diphosphates (NDPs) and nucleoside triphosphates (NTPs) in various tissues of the gastrointestinal tract [1]. Regarding eATP and eADP hydrolysis, ecto-NTPDase-1 hydrolyses both nucleotides at similar rates, while ecto-NTPDase-2 has a high preference for eATP over eADP and ecto-NTPDase3 is a functional intermediate which preferably hydrolyses eATP [14].
Extracellular ATP can be also metabolized by ecto-kinases, with ecto-adenylate kinase (Ecto-AK) facilitating the reversible conversion of eADP to eATP and eAMP, and ecto-nucleoside diphosphate kinase (Ecto-NDPK) promoting the exchange of terminal phosphate between extracellular NDPs and NTPs [13]. All these ecto-enzymes, if present and active, should be able to control the concentration of eATP.
Up to now, although some ecto-enzymes have been identified in intestinal cells, no attempts have been made to characterize the dynamic interaction of these membrane proteins on eATP regulation of intestinal cells. In this study, we aimed to characterize iATP release and eATP recycling by ecto-enzymes, contributing to the regulation of eATP concentration ([eATP]) in Caco-2 cell line. The Caco-2 cells derive from colorectal adenocarcinoma and easily differentiate into cells exhibiting the morphology and function of enterocytes, the absorptive cells of the small intestine [18]. The experimental studies on eATP dynamics in polarized and non-polarized Caco-2 were complemented with a mathematical model quantifying the complex relationship among the different processes contributing to [eATP] regulation. Our results provide a quantitative description of the eATP dynamics of human intestinal epithelial cells.

Results
In this section we show experimental results on eATP kinetics of non-polarized and polarized Caco-2 cells. To understand the dynamics of the different processes contributing to [eATP] regulation, a mathematical model was fitted to experimental data, and predictions were made. Finally, for a comparative purpose, we show results of a few experiments made on epithelial cells obtained from intestinal surgical pieces.

Non-polarized Caco-2 cells
2.1.1. eATP kinetics after hypotonic shock. The kinetics of eATP accumulation, i.e., eATP kinetics, results from the dynamic balance between iATP release mechanisms and the activities of ecto-enzymes capable of degrading and/or synthetizing eATP. As a first step towards the characterization of eATP kinetics, iATP release was triggered by exposing Caco-2 cells to hypotonic media (Fig 1A-1D). Hypotonic swelling is a stimulus that influences the uptake of nutrients by epithelial cells [19] and represents an inducer of iATP release in most cell types and tissues [8].
Under unstimulated conditions, [eATP] remained stable. Whereas addition of isotonic medium triggered a slight increase of [eATP], hypotonic media (100-180 mOsm) activated a stronger iATP release with different kinetics according to the osmotic gradient imposed ( Fig  1A-1C). As shown in Fig 1D, [eATP] increased non-linearly with the magnitude of the hypotonic stimulus.
The experimental [iATP] amounted to 1.81 mM. By comparing [iATP] with [eATP] along eATP kinetics, it was possible to estimate the energy cost of iATP release. Calculations were made for cells exposed to isotonic or 180 mOsm media, two conditions where no lysis was detected [17]. During the isotonic shock, representing a mechanical stimulus in the absence of osmotic gradient, [eATP] amounted to 0.33% of [iATP], while under 180 mOsm this figure amounted to 3.6%. Thus, the energy cost of eATP production by iATP efflux was very small (see section 4.9 for further details). No iADP release was detected in the 180 mOsm stimulus (S1 Fig).
In our previous work, we showed that ecto-nucleotidases present in Caco-2 cells catalyse significant rates of eATP hydrolysis, leading to eADP accumulation [17]. In principle, the resulting accumulated eADP could be used by the potential presence of ecto-kinases like ecto-AK and ecto-NDPK, present in several cell types, to synthetize eATP. Thus, in the following experiments the activities of ecto-AK and ecto-NDPK were assessed by quantifying eATP kinetics under different conditions. from Caco-2 cells after a hypotonic shock was quantified by luminometry performed at room temperature. (A-C) Cells were maintained in isotonic medium and, at the times indicated by the arrow, were exposed to isotonic medium (grey) or to hypotonic media (blue) of 180 mOsm (A), 150 mOsm (B) and 100 mOsm (C). Results are expressed as means of [eATP] from 4, 3 and 2 independent experiments run in triplicate for the 180, 150 a 100 mOsm experiments, respectively. (D) Increases in [eATP] from data in A-C were evaluated as ΔATP, i.e., the difference between [eATP] at 30 minutes post-stimulus and basal [eATP]. Cells were exposed to 300 mOsm (light blue bars), 180 mOsm (blue bars), 150 mOsm (dark blue bars) and 100 mOsm (grey bars). Bars

Ecto-NDPK activity in Caco-2 cells.
NDPK catalyses the transfer of a γ-phosphate from NTP to NDP. Thus, in the presence of eADP and a given eNTP, the following reaction: eADP + eNTP $ eATP + eNDP leads to eATP synthesis when eADP is phosphorylated by NDPK.
Accordingly, incubation of cells with 100 μM eCTP at different [eADP] (3-12 μM) resulted in the rapid synthesis of eATP ( Fig 3A). Maximal [eATP] values were obtained 30 minutes after the addition of substrates (Fig 3A). The experiments were conducted in the presence of 10 μM Ap5A to rule out any contribution of ecto-AK to the observed eATP kinetics.
In separate experiments, addition of increasing [eUTP] (1-100 μM) without the addition of exogenous eADP (only endogenous eADP present), resulted in a concentration-dependent increase of [eATP] (Fig 3C). Because this increase was abolished by 5 mM eUDP (S2 Fig), we hypothesized that eATP synthesis was due to ecto-NDPK activity using exogenous eUTP and endogenous eADP. This is because there is a basal eADP concentration in the extracellular media of 0.77 ± 0.47 μM eADP/mg protein (S3 Fig). A similar experiment using 100 μM eGTP, instead of eUTP, provided qualitatively similar results (Fig 3D). Overall results showed a functional ecto-NDPK activity capable of synthetizing eATP from different γ-phosphate donors (eCTP, eUTP and eGTP) in the presence of endogenous and exogenous eADP.

Modelling eATP kinetics of non-polarized Caco-2 cells.
Caco-2 cells regulate eATP kinetics by iATP release, eATP synthesis by the activities of ecto-AK and ecto-NDPK (as shown in this study), and hydrolysis by ecto-nucleotidases [17]. These processes are active simultaneously when measuring the eATP dynamics in Caco-2 cells, except when a specific inhibitor was added (like Ap5A in the experiments of Fig 3A and 3B). Thus, to quantify the individual contribution of these processes to eATP kinetics, we built a mathematical model that was then fitted to experimental data. Model parameters contain the kinetic information of each enzyme, allowing to assess the individual contribution of ecto-enzymes to eATP dynamics.
A scheme of the model is depicted in Fig 4A. In the model, [eATP] can increase by iATP release, by lytic and by non-lytic mechanisms. In addition, [eATP] can be modulated by the activities of ecto-ATPases, ecto-AK and ecto-NDPK. The model provides functions describing each of the fluxes involved in transport and metabolism of extracellular nucleotides (see S1 Table and section 4.13.1). Fitting the model to the experimental eATP kinetics under the different conditions allowed to obtain the best-fit values for the parameters of these functions (S1 Table). In that way the contributions of each flux to eATP kinetics were quantified, and several predictions were made.

iATP release.
For experiments under iso-and hypotonic media, the model found a good fit to experimental data (continuous lines in Fig 4B), thus allowing to predict the rate of iATP efflux (J ATP ) over time ( Fig 4C). J ATP was rapid and transient in nature, leading to a 12-fold increase of [eATP] to a maximum in less than 2 seconds under the 180 mOsm shock, followed by rapid inactivation. The magnitude of the J ATP peak depended on the osmotic gradient imposed. Inactivation of J ATP was observed under conditions where no lysis was detected (isotonic and 180 mOsm media). On the other hand, a lytic flux (J L ) explains the continuous increase of [eATP] at 100 mOsm (Figs 1C and 4B).

Ecto-enzymes.
Another factor shaping eATP kinetics is eATP hydrolysis by ecto-ATPase activity. We have previously observed that, in intact non-polarized Caco-2 cells, ecto-ATPase activity follows a linear function of micromolar [eATP] [17]. Thus, following a stimulus promoting iATP release, any increase of [eATP] should be at least partially counterbalanced by an increase of ecto-ATPase activity.
Model predictions made at 180 mOsm show that the initial peak of [eATP] increase due to J ATP is about 8-fold higher than the rate of eATP hydrolysis, i.e., J ATP was 1.2 μM iATP/min/ mg of protein ( Fig 4C) and eATP hydrolysis was 0.15 μM eATP/min/mg of protein at 1.5 μM eATP (S1 Table). Thus, during the first seconds of [eATP] increase, eATP kinetics was mainly governed by iATP release. At later times, however, the J ATP inactivated, and the ecto-ATPase activity progressively gained importance in controlling [eATP]. This is illustrated by modelling a change in the amount of ecto-ATPase over a wide range, showing that a 5-fold increase of ecto-ATPase activity could lead to rapid decay of [eATP], while a 5-fold decrease would prolong high levels of [eATP] over the entire incubation period ( Fig 5A). However, a similar procedure, i.e, increasing or decreasing 5 times the activity of ecto-AK, had no influence on the [eATP] during the hypotonic shock (not shown). This can be attributed to the sigmoidal kinetics of ecto-AK, whose activity is very low below 3 μM eADP, but significantly higher above that concentration ( Fig 5D). Thus, ecto-AK might influence eATP kinetics only when [eADP] is sufficiently high. Fig 5B shows a simulation where the initial [eADP] was raised up to 3 μM. At 3 μM eADP, eATP degradation was comparable to eATP synthesis by ecto-AK, indicating that ecto-AK can counterbalance ecto-ATPase activity. Note that a 180 mOsm hypoosmotic  S1 Table for further details). Shadows behind the lines in panels A, B and C represent the uncertainty of the prediction calculated as indicated in section 4.13. Shadows behind the lines in panel D represent the interval ± the standard error of enzyme activity, calculated using the standard error of kinetic parameters of ecto-ATPase and ecto-ADPase activities.
Another factor to consider is ecto-ADPase activity. We have previously shown that Caco-2 cells displays high ecto-ATPase but very low ecto-ADPase activity [17]. Nevertheless, a hypothetical increase of ecto-ADPase activity could negatively modulate ecto-AK activity. For example, an increase of 5-and 12-fold of ecto-ADPase activity would result in a 17% and 33% decrease in the [eATP] production respectively, at 6 μM eADP (Fig 5C).
Model predictions showed above implied that the expression of ecto-AK in Caco-2 cells may have an important role in eATP kinetics. To assess this hypothesis, we compared ecto-ATPase, ecto-ADPase and ecto-AK activities as a function of their respective substrate's concentrations, that is, [eATP] for ecto-ATPase and [eADP] for ecto-ADPase and ecto-AK ( Fig  5D). In Fig 5D, symbols of ecto-AK activities represent the initial velocities for eATP synthesis as a function of [eADP] calculated from experimental data shown in Fig 2A, and the continuous line represents the fit to data of the ecto-AK function included in the model (details in S1 Table and in the work of Sheng and collaborators [22]). The ecto-ATPase and ecto-ADPase activities are predictions made from data of our previous work [17]. Ecto-ATPase displayed the highest rate of the three reactions. On the other hand, although at low [eADP], ecto-AK and ecto-ADPase activities are similar and have relatively low values, the sigmoidal kinetics of ecto-AK allows a strong activity increase as [eADP] is raised, thus reaching activity levels well above those of ecto-ADPase activity ( Fig 5D).
Finally, in the presence of non-adenosine nucleotides, the influence of ecto-NDPK on eATP dynamics was assessed and analysed. Caco-2 cells synthetized eATP by ecto-NDPK activity in the presence of eCTP, eUTP and eGTP as NTP donors, and exogenous and endogenous eADP (Fig 3A-3D).
The model found a good fit to the experimental eATP kinetics in the presence of 100 μM eCTP and different [eADP] (Fig 6A). Model predictions of ecto-NDPK activity at different [eADP] agreed well with initial velocities of experimental ecto-NDPK activities shown in Fig 3A  (Fig 6B). We also studied the effect of eUTP addition without the addition of exogenous eADP  Fig 6D, 6E and 6F). The resulting elevated [eATP] activates ecto-ATPase activity, while ecto-NDPK decreases deeply because its substrates (eUTP and eADP) are consumed by ecto-NTPase activity and by ecto-NDPK activity itself. A balance is then established between ecto-NDPK and ecto-ATPase activities in stage 2, where [eATP] is transiently stable. Finally in stage 3, [eUTP] continues decreasing, leading to a high ecto-ATPase/ecto-NDPK activities ratio, causing [eATP] to decrease and [eADP] to rise again ( Fig 6E).

eATP regulation in polarized Caco-2 cells
Because several reports showed differential activities of enzymes and transporters at each side of polarized epithelia [23,24], we speculated that [eATP] regulation might be different at the apical and basolateral sides of polarized Caco-2 monolayers.
We then used polarized Caco-2 cells to test the effect of hypotonic shock on iATP release and resulting eATP kinetics at the apical and basolateral sides. Similarly to the procedure employed for non-polarized cells, we fitted the model shown in Fig 4A to the experimental data to understand quantitatively the mechanisms involved in [eATP] regulation in differentiated monolayers of Caco-2 cells.
Experimental results show that, following a 180 mOsm hypotonic shock, [eATP] increased at both sides of the monolayers, with qualitatively different kinetics. While at both sides the initial rate of [eATP] increase was fast, apical eATP kinetics achieved a maximum at 1.5 minutes, followed by a rapid decay. This was not observed in the basolateral domain, where [eATP] continued increasing at a progressively lower rate, and a very slow [eATP] decay was observed only after 20 minutes (Fig 7A).
The two different eATP kinetics suggested different activities of ecto-enzymes present at both sides of the monolayers. Therefore, we determined the activities of ecto-ATPase, ecto-AK and ecto-NDPK.
For assessing ecto-ATPase activity, polarized Caco-2 cells were exposed to various [eATP] (0.2-7 μM) and eATP hydrolysis was estimated by quantifying [eATP] decay rates (S5 Fig).  The initial rate values of [eATP] decay were used to calculate ecto-ATPase activity at each [eATP], so as to build a substrate curve (Fig 7B). Linear fitting to experimental data showed that ecto-ATPase activity was 4-fold higher in the apical than in the basolateral domain.
To assess ecto-AK activity, Caco-2 cells were exposed to 12 μM eADP at the basolateral or apical domains. In the apical domain, [eATP] increased rapidly to a maximum, followed by a rapid decay towards pre-stimulated levels, while basolateral [eATP] increased steadily at a lower rate (Fig 7C). Initial velocity estimations showed that ecto-AK activity was significantly higher in the apical than in the basolateral compartment (Fig 7E).
Ecto-NDPK activity was quantified using polarized cells exposed to 100 μM eCTP plus 12 μM eADP in the basal and apical domains. Experiments were run in the presence of 10 μM Ap5A to block ecto-AK activity. Production of eATP by ecto-NDPK was much higher than that observed under conditions used to measure ecto-AK activity, though the domain specific pattern of eATP kinetics was similar when assessing the two ecto-kinases, i.e., a biphasic pattern in the apical domain, and a steady [eATP] increase, at a lower rate, in the basal domain ( Fig 7D). The initial velocity of ecto-NDPK was higher in the apical than in the basolateral domain although differences were not significant (Fig 7F, p Table). Results indicated that the ecto-NDPK maximal activity in the apical compartment is a slightly higher than that of the non-polarized cells and significantly higher than that of the basolateral compartment. On the other hand, the ecto-AK maximal activity is significantly higher compared with the basolateral compartment or the non-polarized cells. Thus, the differences between the apical and basolateral eATP dynamics can be explained by an increase or decrease in ecto-enzymes activities.
Altogether experimental results showed significantly higher activities of the ecto-enzymes (ecto-ATPase, ecto-AK and ecto-NPDK) in the apical, as compared to the basolateral domain ( Fig 7G).

Ecto-AK and ecto-NDPK are active in human primary small intestinal epithelial cells
Having characterized ecto-AK and ecto-NDPK activities of Caco-2 cells, we wondered whether these ecto-enzymes would be functional in IECs extracted from human small intestine. Accordingly, we used samples obtained from small intestine biopsies from healthy donors (Fig 8A and 8B).

Discussion
Intestinal epithelial cells can release iATP and express several ecto-enzymes capable of regulating the amount and metabolism of eATP at the cell surface. The main goal of this study was to characterize quantitatively the dynamic interplay of iATP release, eATP hydrolysis and eATP synthesis contributing to the dynamic regulation of [eATP] in Caco-2 cells. Special emphasis was given to the role of ecto-kinases promoting eATP production under different conditions.
Since Caco-2 cells undergo spontaneous enterocytic differentiation in culture, we decided to first approach the complexity of eATP regulation using the relatively simpler non-polarized cell model, and later extend the study to fully differentiated cells. These form apical and basolateral poles where morphological and biochemical features are segregated [23].
When exposed to hypotonicity, non-polarized Caco-2 cells triggered a strong iATP efflux that rapidly inactivated, leading to low μM [eATP] accumulation. A number of studies have confirmed that such micromolar [eATP] are capable of activating P2 receptors with high affinity for that nucleotide, such as P2Y2, P2Y11 and almost all P2X receptors [25]. In Caco-2 cells, eATP dose dependently activates P2Y receptors involved in the activation of MAPK cascades and transcription factors that promote cell proliferation [26,27], while higher [eATP] can induce apoptosis via P2X7 receptor [3].
In principle, purinergic activation by eATP should be transient, due to the presence of ectonucleotidases, the activities of which promotes strong eATP hydrolysis in Caco-2 cells [17]. Accordingly, our results show that hypotonicity induced iATP release and concomitant eATP accumulation, where [eATP] decay was accelerated by constitutive ecto-ATPase activity. This decay was even higher for a model predicted upregulation of eATP hydrolysis by one or more ecto-nucleotidases, as occurs in various cells and tissues during pathogen infection [28], cell differentiation [29] or tumorigenesis [30].
The above results imply that iATP release and eATP hydrolysis constitute two opposing fluxes shaping eATP kinetics of Caco-2 cells. However, the presence of ecto-kinases found in this study suggest that the dynamic regulation of [eATP] should also take the activities of these enzymes into account.
In this respect, addition of exogenous eADP to Caco-2 cells dose dependently increased [eATP]. The fact that eATP synthesis was almost fully blunted by Ap5A, an AK blocker that does not permeate intact cells, suggested the presence of a functional ecto-AK. Results of the mathematical model allowed to envisage the contribution of ecto-AK to eATP kinetics. In the absence of exogenous eADP, the contribution of ecto-AK to eATP kinetics was negligible, so that [eATP] depended mainly on the balance between the rates of iATP release and eATP hydrolysis. This is due to the low endogenous [eADP] present under the experimental conditions. However, due to the sigmoidal nature of the AK reaction, model predictions show that increasing [eADP] in the low micromolar range, suffices to promote significant eATP synthesis by ecto-AK, upregulating eATP kinetics. Thus, under certain conditions, e.g., when cell leak intracellular ADP (iADP) or eADP is supplied paracrinally by other cell types, eATP synthesis by ecto-AK of Caco-2 cells will transiently stabilize [eATP] levels, thereby favouring propagation of eATP-dependent purinergic signalling. A similar stabilizing role of ecto-AK on [eATP] has been proposed for HT29 cells, lung epithelial cells and lymphocytes [14,15,31].
Modelling shows that ecto-ADPase activity, which facilitates eADP degradation, may compete with ecto-AK for the available eADP. However, Caco-2 cells -as HT29 cells [15]-displayed a relative low ecto-ADPase activity, in agreement with the presence of a functional ecto-NTPDase 2 in both cell types [15,17], and in addition the intrinsic sigmoidal nature of ecto-AK activity makes ecto-AK more sensitive to [eADP] than ecto-ADPase. Another consequence of ecto-AK activation relates to P1 signalling, since activity of this enzyme will provide eAMP from eADP for further hydrolysis to adenosine by ecto-5'NT present in Caco-2 cells [17], finally leading to extracellular adenosine accumulation.
Our model predictions show how increasing [eADP] in the low μM range might lead to substantial adenosine accumulation, which may engage 4 different P1 receptors [32]. The consequences of P1 signalling on proliferation of Caco-2 cells and several other intestinal epithelial cell lines have been studied before [33]. In general, the balance between P1 and P2 receptors on epithelial cells regulate intestinal secretion [34][35][36][37] and absorption [38,39]; responses triggered by the P2 receptor stimulation by eATP and other nucleotides are sometimes counteracted by P1 receptor stimulation by adenosine, though the potential role of ecto-AK was not considered in this context.
Another factor affecting eATP kinetics is ecto-NDPK. Activity of this enzyme was detected in many cells and tissues such as astrocytoma cells [40], endothelial cells [41,42], lymphocytes [41], keratinocytes [43] and hepatocytes [44]. In general, ecto-NDPK will primarily serve to transfer phosphate groups between different extracellular nucleotides and thus potentially alter the pattern of P2 receptor activation. This is especially important since P2 receptor subtypes are differentially selective for adenine and uridine eNDPs and eNTPs [45,46].
Our results show that ecto-NDPK can use eCTP, eGTP and eUTP to phosphorylate eADP to eATP. As model predictions show, activities of ecto-NDPK (promoting eATP synthesis from eUTP and eADP) and ecto-nucleotidase (promoting eATP and eUTP hydrolysis) change in opposite directions to transiently stabilize [eATP].
Results analysed above show that, in non-polarized Caco-2 cells, [eATP] can increase by iATP release and ecto-kinase mediated eATP synthesis and decrease by ecto-nucleotidases mediated by eATP hydrolysis.
Next, we studied eATP dynamics of polarized Caco-2 cells. These cells differentiate spontaneously into polarized cells, with apical and basolateral domains exhibiting morphological and biochemical features of small intestine enterocytes [23,47]. In particular, the Caco-2 polarized phenotype is characterized by high levels of hydrolases typically associated with the brush border membrane. The fact that in a variety of epithelia several ecto-nucleotidases and ecto-phosphatases preferentially -but not exclusively-locate in the apical domain [48][49][50], anticipated a different eATP regulation at both poles of Caco-2 cells.
Accordingly, hypotonically induced eATP kinetics had a faster resolution and was more effectively regulated at the apical, than at the basolateral side, a result in agreement with the observed higher apical (than basolateral) ecto-ATPase activity measured in this study. This is in agreement with several reports using intestinal epithelial cell from murine models and human intestinal cell lines, showing that various isoforms of ecto-NTPDases, ecto-phosphatases and ecto-NPPases are preferentially located in the apical domain [50].
The mechanism mediating iATP release at both sides of the polarized Caco-2 cell monolayer requires further investigation. We previously showed that iATP release in Caco-2 cells challenged by adrenergic stimulation or the presence of bacteria is reduced by treatment with carbenoxolone, a blocker of conductive iATP efflux [51]. Since the molecular mechanisms involved in iATP release may depend on the stimulus, further investigations will be conducted to clarify this topic.
A qualitatively similar pattern was observed for ecto-AK and ecto-NDPK of Caco-2 cells, in that apical activities were much higher. Interestingly, the model describing eATP dynamics of non-polarized cells could be successfully fitted to eATP kinetics on each of the polarized domains, thus allowing to calculate the consequences of ectoenzymes sorting on eATP regulation.
The results obtained with primary human IECs suggest that our model could be employed to explain the eATP kinetics in these cells, given the presence of both ecto-AK and ecto-NDPK. However, further studies would be required to adapt the Caco-2 model to IECs. These studies may need to account for bacterial periplasmic ATPases capable of hydrolyzing eATP, as we and others have shown that commensal bacteria can express periplasmic nucleotidases and release ATP into the intestinal lumen [51,52]. In this context, the model presented here may be taken as a starting point to progressively add other processes affecting [eATP] regulation in vivo.
The fact that the apical domain exhibited a higher turnover of extracellular nucleotides, leading to higher eATP regulation may have adaptive value, considering that iATP release is a common response of epithelial intestinal cells to enteric pathogens [53]. Extracellular ATP may then act as a danger signal controlling a variety of purinergic responses aimed at defending the organism from a variety of pathogens and their toxins present in the intestinal lumen.

Ethics statement
The protocol for handling human samples was approved by the Institutional Review Board of the Favaloro Foundation University Hospital (DDI [1587] 0621) and has been performed in accordance with the ethical standards laid down in the declarations of Helsinki and Istanbul. Informed written consent was obtained from donors.

Solutions
In the experiments to measure [eATP] by luminometry (section 4.5), cells were incubated with isotonic buffer called isosmotic DPBS (300 mOsm) containing: 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 1.5 mM KH 2 PO 4 and 8 mM Na 2 HPO 4 , pH 7.4 at 37˚C (assay medium). When applying a hypotonic shock to cells, the medium was changed for other containing the same components but with a lower NaCl concentration. Thus, DPBS with 100, 150 and 180 mOsm were prepared. The osmolarity of all media was measured with a vapor pressure osmometer (5100B, Lugan, USA).

Polarisation of Caco-2 cells.
For preparation of polarized Caco-2 monolayers, cells were seeded in permeable supports (inserts) made of polyester (Transwell; 0.1 μm pore size, 1.12 cm 2 cell growth area; Jet Biofil, China) in 12-well plates at a density of 3 × 10 4 cells/0.5 mL per insert. The medium was changed after 3 days, and then after every 3 or 4 days. The polarized Caco-2 monolayers were used for experiments after the transepithelial electrical resistance reached a plateau (approximately 21 days after seeding). In polarized and non-polarized cultures contamination (including Mycoplasma) was routinely tested.

Human Intestinal Epithelial Cells (IECs) isolation
IECs were isolated from ileum biopsies collected from healthy volunteers who were endoscopically evaluated for colon cancer (N = 3) at the Favaloro Foundation University Hospital. Samples of non-tumoral, non-injured intestinal biopsies were collected and transported in ice-cold Hanks's balanced salt solution (HBSS) for immediate processing. The biopsies were incubated in 5 mM ethylenediaminetetra-acetic acid (EDTA) and 1.5 mM dithiothreitol HBSS with agitation for 25-30 minutes at room temperature to obtain IECs. Cells were pelleted, re-suspended in DPBS and used immediately.

ATP measurements
The [eATP] of non-polarized Caco-2, polarized Caco-2 monolayers or IECs was measured using the firefly luciferase reaction (EC 1.13.12.7, Sigma-Aldrich, St Louis, MO, USA), which catalyses the oxidation of D-luciferin in the presence of ATP to produce light [54]. As described below, using this method it was possible to determine eATP kinetics, the iATP content and the activities of ecto-enzymes. Before the experiments, the cells were washed two times with the assay medium (isosmotic DPBS with or without Pi).
In this work, the cells' medium was substituted by the assay medium before any measurement, therefore exoenzymes (enzymes released to extracellular medium not bound to the membrane) were removed and only ecto-enzymes (membrane bound extracellular enzymes) were investigated.

eATP kinetics of non-polarized Caco-2 and IECs
Non-polarized Caco-2 cells and IECs were seeded on glass coverslips. Under all conditions cells were mounted in the assay chamber of a custom-built luminometer, as previously described [55]. Because luciferase activity at 37˚C is only 10% of that observed at 20˚C [56], to maintain full luciferase activity, [eATP] measurements were performed at room temperature. The setup allowed continuous measurements of [eATP] by the luciferin-luciferase reaction.
A calibration curve was used to transform the time course of light emission into [eATP] versus time. Increasing [eATP] from 13 to 1000 nM were sequentially added to the assay medium from a stock solution of pure ATP dissolved in isosmotic or hypotonic medium, according to the experiment. Calibration curves displayed a linear relationship within the range tested. After each experiment, cells were lysed with a solution containing 1 mM PMSF and 0.1% of Triton X-100 and the protein contents of each sample were quantified [57]. Results were expressed as [eATP] at every time point of a kinetics curve denoted as "eATP kinetics", with [eATP] expressed as μM of eATP/mg protein in a final assay volume of 100 μL.

eATP kinetics of polarized monolayers
Polarized Caco-2 cells monolayers were placed in the insert physically separating an apical and a basolateral compartment. Detection of eATP was performed separately on either side, by adding the luciferin-luciferase mixture in one compartment (apical or basolateral) and adding isosmotic DPBS to the other side. In preliminary experiments, we observed that the luciferinluciferase mix added in one compartment did not cross the monolayer into the other compartment. Thus, luminescence registered when measuring the [eATP] in one compartment was not contaminated by light from the other compartment due to luciferin-luciferase leakage.
When an hypoosmotic shock was applied, a luciferin-luciferase mix in DPBS with an osmolarity of 180 mOsm was added to the compartment of interest while, isosmotic DPBS was added to the other side.

Activities of ecto-enzymes
Ecto-ATPase, ecto-AK and ecto-NDPK activities of intact cells were measured by luminometry (section 4.5). Ecto-nucleotidase activities were measured by measuring the inorganic phosphate (Pi) release.
Activities measured in section 4.8.1 were expressed as μM of eATP hydrolysed per minute, normalized by the cell protein mass in the experimental sample (μM of eATP /mg protein/ min). Results from experiments explained in sections 4.8.2 and 4.8.3 were expressed as μM of eATP synthetized per minute, normalized by the cell protein mass in the experimental sample (μM of eATP /mg protein/min). Activities measured in section 4.8.4 were expressed as μM of inorganic phosphate released per minute, normalized by the cell protein mass in the experimental sample (μM of Pi /mg protein/min).

Intracellular ATP measurements
Caco-2 (0-30,000 cells) were laid on coverslips, incubated with 45 μL of luciferin-luciferase reaction mix for 5 minutes and subsequently permeabilized with digitonin (1.6 mg/mL final concentration). Light emission was transformed into [eATP] as a function of time as indicated in section 4.6. After considering the total volume occupied by Caco-2 present in the chamber, and the relative solvent cell volume (3.66 μl per mg of protein) [59], [iATP] was calculated in mM. To calculate the % of iATP release, the following equation was employed:  [59]) and diving by the assay volume (0.1 mL).

Extracellular ADP measurements
For detection of eADP of intact Caco-2 cells, 3 U/100 μL of pyruvate kinase and 100 μM PEP were added to the luciferin-luciferase mix. Using PEP as a substrate, pyruvate kinase promotes the stoichiometric conversion of eADP into eATP [60]. The resulting eATP was then measured by light emission using the luciferin-luciferase procedure described above.

Data analysis
Statistical significance was determined using the non-parametric Mann-Whitney test. Data were analyzed and graphically represented using GraphPad Prism software v5.0 (Graph Pad Software, San Diego, CA, USA). Each independent experiment was carried out in an independent cell culture or tissue sample in a different day.

Initial velocity estimation
To measure the initial velocity of Ecto-AK or Ecto-NDPK, the eATP dynamics were measured as indicated in section 4.8.2 and 4.8.3. Only the values of [eATP] obtained during the first 5 minutes after substrates addition were considered for further analysis. The following equation was fitted to experimental data: where A and k are parameters, whose value are optimized to achieve a good fitting of Eq 2 to experimental data. The initial velocity is the derivative of [eATP] as a function of time at time 0 (the time when substrates were added). Thus, the initial velocity was calculated by multiplying the value of A by the value of k.

Mathematical modelling
Chemical models of extracellular nucleotides were built using COPASI (Complex Pathway Simulator) software in version 4.29 (source: https://copasi.org/) [61]. Parameter optimization was performed using COPASI "parameter estimation function" with Hooke & Jeeves (50 iteration steps, 10 −5 tolerance and 0.2 rho factor), Levenberg-Marquardt (2000 iteration steps, 10 −6 tolerance), or Evolutionary programming (200 generations with a 20 population size) as optimization methods. An initial guess of the parameter value was proposed based on literature data for each kinetic step. A detailed description of the models employed in this work can be found in S1 and S2 Tables. Parameters obtained from the model fitting are expressed as the best value ± standard deviation. The COPASI and SBML files of the models described in section 4.13.1 and 4.13.2 can be found in the data repository (see data availability statement). When performing time course simulations, we used the deterministic LSODA method with default parameters in COPASI version 4.29. Specifically, we set the relative tolerance to 10 −6 , the absolute tolerance to 10 −12 , and allowed for a maximum of 10 5 internal steps without a limit on maximal step size.
Model prediction uncertainties were calculated using the parameter scan task in COPASI, which explores the parameter space within the interval given by the parameter best value ± the standard error (S1 Table). Four parameter values were considered within the range of the parameter best value ± the standard error, one at the lowest value of the interval, one at the highest and two in the middle. Simulations were performed by testing all possible combinations of the selected parameter values. The shaded regions depicted in Figs 5 and 6 correspond to the area that includes all simulations results obtained by varying the parameters values. In Fig 5, the prediction uncertainty was calculated by simulating the eATP kinetics while varying the parameters K ATP , K ADP , V max Ecto-AMPase and Ftr AK . These are the kinetic parameters that control eATP kinetics under the hypotonic stimulus. In Fig 6D, 6E and 6F, the prediction uncertainty was calculated by simulating the nucleotides kinetics by varying the parameters K ATP , K mAD , K mUTP V max NDPK and K NTPase .

A model of purinergic homeostasis in non-polarized Caco-2 cells.
To explain the experimental observations, a data driven mathematical model was created (depicted in Fig  4A). The model has 7 reactions to explain the chemical fluxes of transformations or transport of extracellular nucleotides in Caco-2 cells: J ATP , J Ecto-ATPase , J Ecto-ADPase , J Ecto-AMPase , J Ecto-AK , J Ecto-NDPK and J Ecto-NTPDase . A detailed description of each flux, its mathematical description and parameters can be found in S1 Table. In the model, the concentration of each species as a function of time was calculated from the following differential equations: Note that in the equations J Ecto-AK and J Ecto-NDPK were considered in the direction of eATP consumption, i.e., eAMP + eATP $ 2 eADP for J Ecto-AK and eNDP + eATP $ eNTP + eADP for J Ecto-NDPK . The model was written in COPASI 4.29 and was fitted simultaneously to all experimental data shown in Figs 1A, 1C, 2A, 3A and 3B. The fitting of the model to experimental data can be seen in Figs 4B, 5C, 6A and 6C as red lines.
Some kinetic parameters of the enzymes catalyzing the reactions were obtained from the literature. Parameters from the J ecto-ATPase and J ecto-ADPase were obtained from our previous work [17]. The V max of the J ecto-AMPase reaction was obtained from our previous work [17], while the K m was obtained from the work of Navarro et al. [62]. Kinetic parameters of the J ecto-AK activity were obtained from the work of Sheng et al [22]. The equilibrium constant (K eq ) and the affinity for ATP (K mAT ) of the J ecto-NDPK were obtained from the work of Garces and Cleland [63]. The affinity constants for product inhibition in J ecto-NDPK (K iNDP and K iADP ) were estimated from the work from Lascu et Gonin [64]. The rest of the model parameters were obtained from model fitting to experimental data (see S1 and S2 Tables for more details). The shape of the J ATP flux as a function of time was modeled based on findings of a previous work from our group [65].

A model of purinergic homeostasis in polarized Caco-2 cells.
The model fitted to experimental data from the apical and basolateral compartments data is the same model indicated in section 4.13.1, although the parameters of some reactions were fitted again (S2 Table). The J ATP expression for the 180 mOsm hypotonic shock in the polarized cells was different from the one employed on non-polarized cells (S2 Table). The mathematical expressions of the other 6 reactions were not modified. Four parameters were refitted to the data to account for differences in the ecto-ADPase, ecto-AK and ecto-NDPK activities after polarization (values can be found in S2 Table). Moreover, in the case of ecto-NTPDase, the eCTP hydrolysis could not be neglected in the apical compartment and was necessary to achieve a good fit to experimental data. In contrast the eCTP hydrolysis could be avoided in the basolateral compartment without affecting model fitting. This suggest that the ecto-CTPase activity is greater in the apical than in the basolateral compartment, in agreement with the increased activity of other enzymes on the apical side.
The differential equations for [eCTP] and [eCDP] are modified in the apical side model to account for the eCTP hydrolysis: The models for the apical and basolateral compartments were written in COPASI 4.29 and fitted to experimental data shown in Fig 7A, 7C Table  (for the apical and basolateral compartments) and in S1 Table (for the non-polarized cells). The ecto-AK V max (B) was calculated from the model parameters using the following formula: Ftr AK k À 2 k 1 k À 2 þk 1 , where the Ftr AK was obtained from the model fitting (S2 Table for the apical and basolateral compartments and S1 Table for the non-polarized cells). The k -2 and k 1 parameters value can be found in S1 Table. (TIF) S1 Table. Mathematical model of eATP regulation in non-polarized Caco-2 cells. Numerical values of constants were normalized by the protein cell mass in the experiments (M cell ), measured by the Bradford method (section 4.7 in the manuscript). Parameter fitting and simulations were performed by selecting the average cell mass in the experiments (M cell = 0.2 mg). J L and J NL represent the lytic and non-lytic iATP release respectively upon an osmotic shock.
The value of these terms was 0 before shock application. J leakage represents a constant and small iATP release observed in the absence of any stimulus. The parameter values obtained from the model fitting are expressed as the best value ± standard deviation. (XLSX) S2 Table. A: J NL parameters obtained from fitting to experimental data at 180 mOsm shock in apical or basolateral compartments in polarized cells. The same value of k obs was considered for both compartments. Parameters obtained from the model fitting are expressed as the best value ± standard deviation. B: Parameters obtained from model fitting to experimental in apical or basolateral compartments in polarized cells. The model equations are the same shown in S1 Table, however, some parameters values were fitted again to experimental data from polarized cells. The parameters whose value has changed in comparison with the model of non-polarized cells are shown in this file. The rest of the parameters had the same value for non-polarized cells (shown in S1 Table). The K ADPase and K NTPase (for eCTP) were considered 0 in the basolateral compartment. This does not mean that there is no ecto-ADPase or ecto-NTPase activity in the basolateral side but, they can be neglected in our experimental conditions. Parameters obtained from the model fitting are expressed as the best value ± standard deviation. (XLSX)