Purinergic receptors and regulatory volume decrease ...

Fish Physiol Biochem DOI 10.1007/s10695-012-9653-x

Purinergic receptors and regulatory volume decrease in seabream (Sparus aurata) hepatocytes: a videometric study Agata Torre • Francesca Trischitta Caterina Faggio

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Received: 2 February 2012 / Accepted: 30 April 2012 Ó Springer Science+Business Media B.V. 2012

Abstract The response of isolated hepatocytes of Sparus aurata to hypotonic stress was studied by the aid of videometric methods with the aim to investigate the possible involvement of ATP in the regulatory volume decrease (RVD). This study confirms our previous observations showing the ability of these cells to undergo RVD. In addition, it shows that the homeostatic response was inhibited by apyrase, an ATP scavenger, thus suggesting the involvement of extracellular ATP in the RVD response. Experiments performed in the presence of ATPcS or adenosine, agonists of P2 and P1 receptors respectively, and in the presence of suramin or 8-PT, antagonists of P2 and P1 receptors respectively, suggest that ATP exerts its stimulatory effect on the homeostatic response by interacting with P2 receptors. On the other hand, the activation of P1 receptors by ATP metabolites produces opposite effects. In an attempt to clarify the mechanisms involved in ATP release from the cell, we performed some experiments with known inhibitors of the possible mechanisms of regulated ATP release. The results we obtained let us to suppose that the mechanism allowing the exit of ATP from the cell is verapamil sensitive suggesting the involvement of the P-glycoprotein.

A. Torre F. Trischitta (&) C. Faggio Dipartimento di Scienze della Vita ‘‘M. Malpighi’’, Universita` di Messina, Viale Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy e-mail: [email protected]

Keywords Hepatocytes Sparus aurata RVD Purinergic receptors ATP release

Introduction Cell volume regulation is essential for cell survival. Most animal cells exposed to hypotonic conditions show an initial rapid swelling, due to an osmotic influx of water, followed by a tendency to recover the initial size. This regulatory response is called regulatory volume decrease, RVD, and is achieved by a release of organic solutes, such as amino acids and ions through the activation of K? channels and/or anion channels, KCl cotransport, or parallel activation of K?/H? exchange and Cl-/HCO3-exchange followed by a water efflux from the cells (Fugelli and Thoroed 1986; Hoffmann et al. 2009). To avoid excessive alterations of cell volume, however, the cells not only activate transport mechanisms across cell membrane but also utilize metabolism. These mechanisms are triggered by minute alterations of cell volume. They not only serve to readjust cell volume but profoundly modify a wide variety of cellular functions. Thus, cell volume is an integral element within the cellular machinery regulating cellular performance (Lang et al. 1998). Therefore, cell volume during the whole life cycle of a cell represents an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death

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and the regulation of intracellular metabolism. Moreover, it has become increasingly evident that dysfunctional cell volume regulation may significantly contribute to the pathophysiology of several disorders such as liver insufficiency, diabetic ketoacidosis, hypercatabolism, fibrosing disease, sickle cell anemia, and infection (Wehner et al. 2003). Therefore, it is not surprising that many factors can be involved in the activation of volume homeostatic response (Hoffmann et al. 2009). Since the study of Wang et al. (1996), a signaling role of ATP on RVD was suggested for both fish (Ballatori and Boyer 1997; Ollivier et al. 2006b; Pafundo et al. 2004, 2008) and mammals (Feranchak et al. 2000; Roman et al. 1997). ATP is a multifunctional biological molecule that acts not only intracellularly as the primary source of energy for living cells but also extracellularly as a signaling molecule that regulates diverse cellular processes including synaptic transmission, nociception, ion transport, apoptosis, secretion, bladder contraction, vascular tone, immune responses, muscle contraction, cell proliferation, mucociliary clearance, platelet aggregation and neurotransmission, cell swelling (Beigi et al. 1999; Grygorczyk and Hanrahan 1997; Novak 2003; Sorensen and Novak 2001; Yegutkin and Burnstock 2000). ATP is abundant in the cell cytoplasm (3–5 mM) and can be released extracellularly by several mechanisms (Chara et al. 2011; Fitz 2007). Extracellular ATP can act as a potent autocrine/paracrine signal by binding to cell-surface purinergic receptors belonging to two classes of receptors: the P1 and P2 classes (Burnstock 2007). Our previous studies showed that Sparus aurata hepatocytes are able to perform RVD when exposed to hypotonic conditions (Faggio et al. 2011). This study has the aim to investigate, by employing videometric measurement, if the RVD response in seabream hepatocytes depended on extracellular ATP, as suggested for goldfish, trout, and turbot (Ollivier et al. 2006b; Pafundo et al. 2004, 2008), and which possible receptors could be involved in this response.

Materials and methods Isolation of hepatocytes Gilthead sea breams (S. aurata, 60–100 g) obtained from a fish culture in Patti, Messina (Italy), were kept

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in the Centro di Ittiopatologia sperimentale della Sicilia (C.I.S.S.) in 800-l tanks with flowing seawater at 18 °C and fed with dry pellets for fish. Before the experiments, the fish were anesthetized with MS 222. The liver was removed and subsequently hepatocytes were isolated by collagenase digestion methods, which required perfusion of the portal vein as described in Ollivier et al. (2006a). Isolated hepatocytes were maintained in a 370 mOsm/kg isotonic solution corresponding to the extracellular osmolality measured in the serum of this fish by a Fiske osmometer that determines the osmolarity using freezing point depression. This physiological solution contained (millimoles/l): 172 NaCl, 3.4 KCl, 0.8 MgSO4, 1.5 CaCl2, 5 NaHCO3, 0.33 Na2HPO4, 0.44 KH2PO4, 5 glucose and 10 Hepes (pH = 7.4). Cells were maintained under slight agitation for at least 1 h at 18 °C before starting the experiments to re-establish ionic concentrations on either side of the cell membrane. Cell population in which viability, measured by the Trypan blue exclusion method, was less than 85 % post isolation was discarded. To assess the viability of the cells at the end of each experiment, the experimental solution was changed with an isotonic solution containing Trypan blue, and the viability was tested after 10 min preincubation. The experiment was discarded if the viability was affected. The letter n in the figure captions indicates the number of cells observed; however for each experimental condition, at least three preparations were used. Cell size experiments In order to immobilize cells, they were seeded onto the bottom of a thermostated plexiglass chamber (18 °C) coated with poly-L-lysine (0.01 %) and left to adhere for 2 min. Cells were incubated in isotonic solution for 3 min, and then, an osmotic shock (from 370 to 260 mOsm/kg) was performed by substituting the isotonic solution with the hypotonic solution (millimoles/l: 124 NaCl, 3.4 KCl, 0.8 MgSO4, 1.5 CaCl2, 5 NaHCO3, 0.33 Na2HPO4, 0.44 KH2PO4, 5 glucose and 10 Hepes; pH = 7.4). Changes of S. aurata hepatocyte size were assessed by the use of a videometric method (see Faggio et al. 2011). Under each experimental condition, the cell images were recorded at regular time intervals and transferred to the computer in order to trace the cell outline from which the area of


Statistic All data are expressed as mean ± SE. Statistical analyses were performed using the one-way ANOVA test for repeated measures followed by a posteriori comparisons (Bonferroni test). When differences between control and treated cells were compared at a given time point, the two-way ANOVA test for repeated measures was used. Differences were considered significant at p \ 0.05.

Hypotonic

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the sagittal section was calculated with the aid of the ImageJ software. The areas of the cells in every experimental condition (Aexp) were compared to the areas measured in isotonic solution (Ai) at the beginning of the experiments. So the data are reported as relative area Aexp/Ai. Even if we did not measure cell volume, taken the spherical shape of isolated hepatocytes, we supposed that changes of the measured area are paralleled by changes of volume, so sometimes in the text, we indicate the area changes as volume changes. When the drugs were tested, all of which were obtained from Sigma Aldrich (St. Louis, Mo, USA), they were added to both the isotonic (15 min) and hypotonic solutions in the perfusion chamber. 8-PT (8phenyltheophylline), glybenclamide and verapamil were dissolved in DMSO, its final percentage in the chamber was 0.1 %, a solvent concentration that did not alter cell size.

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time (min) Fig. 1 Relative area changes in the isolated hepatocytes exposed to the hypotonic solution; n (number of cells) = 29. The values are means ± SE. * p \ 0.05 versus isotonic condition; ** p \ 0.05 versus maximum swelling

applying the hypotonic shock. In order to verify whether the isotonic perfusion for 18 min could have an incidence on cell volume, we performed preliminary control experiments in which the cells (20 cells, 5 preparation) were exposed to an isotonic perfusion for 18 min before hypotonic shock. The longer isotonic perfusion did not modify neither cell volume nor the following response to the hypotonic shock (not shown) that was identical to that described in Fig. 1. In order to assess the involvement of ATP in the RVD response, the cells, preincubated for 15 min with the ATP scavenger apyrase (3 U/ml), were exposed to the hypotonic solution also containing the drug. As Fig. 2 shows, the hypotonic stress given in the Apyrase

Results

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Figure 1 shows the response of isolated hepatocytes of S. aurata to a rapid change in osmolarity of bathing solution. After having perfused the cells with isotonic solution for 3 min, a rapid change with a hypotonic solution was performed. The cells, on continuing exposure to hypotonic conditions, showed a rapid initial swelling reaching its maximal value after 5 min; this enlarged size was maintained for about 20 min after which the cells underwent RVD to approach their initial size, despite the continuous osmotic gradient. In the RVD experiments performed in the presence of drugs, the cells were perfused for 3 min in isotonic solution and thereafter were incubated with isotonic solution containing the tested drug for 15 min before

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time (min) Fig. 2 Effect on RVD of apyrase addition (3 U/ml) to the hypotonic solution. Apyrase was added 15 min prior to exposure to the hypotonic solution which also contained the drug; n (number of cells) = 6. The values are means ± SE. * p \ 0.05 versus isotonic condition

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Fish Physiol Biochem Suramin Hypotonic 1.2 1.15

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presence of the scavenger produced a continuous cell swelling, leading to a size (1.579 ± 0.190) significantly larger (p \ 0.01) than the maximum size observed in the cells exposed to hypotonic saline in its absence (1.112 ± 0.001). Since these results suggested a role of extracellular ATP in the RVD response, we performed some experiments in the presence of agonists and antagonists of P2 and P1 receptors in order to know the membrane target of the nucleotide. Figure 3 shows that when the hypotonic stress was applied in the presence of P2 receptor agonist ATPcS (5 lM), the cells swelled as usually but the onset of the RVD response was more rapid (5 min) than in the control conditions (20 min). In addition, in ATPcS experiments, the cell size reached a significant reduction (p \ 0.05) respect to the maximum swelling after 25 min, while in the control experiment after 35 min. On the contrary, suramin (100 lM), antagonist of P2 receptors, inhibited RVD (Fig. 4). Figure 5 shows that a complete recovery of cell volume after the initial swelling was observed when the cells were bathed with the hypotonic solution containing 8-PT (100 lM), an antagonist of P1 receptors; in addition, the onset of the homeostatic response was more rapid (5 min) than in the control conditions (20 min). On the other hand, the P1 agonist adenosine (5 lM) inhibited the homeostatic response (Fig. 6).

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time (min) Fig. 3 Effect on RVD of the addition of ATPcS (5 lM) to the hypotonic solution. ATPcS was added 15 min prior to exposure to the hypotonic solution which also contained the drug; n (number of cells) = 21. The values are means ± SE. * p \ 0.05 versus isotonic condition; ** p \ 0.05 versus maximum swelling

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Fig. 4 Effect on RVD of the addition of suramin (100 lM) to the hypotonic solution. Suramin was added 15 min prior to exposure to the hypotonic solution which also contained the drug; n (number of cells) = 21. The values are means ± SE. * p \ 0.05 versus isotonic condition

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Fig. 5 Effect on RVD of the addition of 8PT (100 lM) to the hypotonic solution. 8PT was added 15 min prior to exposure to the hypotonic solution which also contained the drug; n (number of cells) = 23. The values are means ± SE. p \ 0.05 ** versus maximum swelling

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In the attempt to establish the mechanism by which ATP is released by the cell, we tested the effect on RVD of a potential inhibitor of ATP release, verapamil (10-4 M). Figure 7a shows that the hypotonic shock in the presence of verapamil produced a continuous cell swelling and no RVD response. When verapamil was tested in the presence of ATPcS, we observed that the cells, after the initial swelling, exhibited a partial recovery of cell volume (Fig. 7b).

Fish Physiol Biochem Adenosine

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time (min) Fig. 6 Effect on RVD of the addition of adenosine (5 lM) to the hypotonic solution. Adenosine was added 15 min prior to exposure to the hypotonic solution which also contained the drug. n (number of cells) = 23. The values are means ± SE. * p \ 0.05 versus isotonic condition

Fig. 8 Effect on RVD of the addition of ATPcS (5 lM) to the Ca2?-free hypotonic solution. ATPcS was added 15 min prior to exposure to the hypotonic solution which also contained the drug; n (number of cells) = 6. The values are means ± SE. * p \ 0.05 versus isotonic condition

The cells did not exhibit RVD when ATPcS was tested in calcium-free hypotonic medium (Fig. 8) or in the presence of 5 9 10-4 M glybenclamide (Fig. 9).

comes from the observation that the addition of the ATP scavenger apyrase to the hypotonic medium inhibited the RVD response (Fig. 2). Such a dependence of RVD on extracellular ATP has been already demonstrated in other teleost hepatocytes such as those of Onchorynchus mykiss (Pafundo et al. 2004), of Scophthalmus maximus (Ollivier et al. 2006b), and of Carassius auratus (Pafundo et al. 2008), thus confirming the original hypothesis of Wang et al. 1996 in a study on rat HTC hepatoma cell indicating ATP as a novel autocrine signal involved in cell volume regulation. It was suggested that extracellular ATP regulates the mechanism responsible of RVD by activating the

Conclusions This study confirms our previous observations (Faggio et al. 2011), suggesting that seabream hepatocytes, when subjected to hypotonic conditions, showed a rapid swelling and thereafter a regulatory volume decrease (Fig. 1). In addition, it suggests that this homeostatic response depends on extracellular ATP. This idea

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time (min) Fig. 7 a Effect on RVD of the addition of verapamil (10-4 M) to the hypotonic solution. Verapamil was added 15 min prior to exposure to the hypotonic solution which also contained the drug: n (number of cells) = 6. The values are means ± SE. * p \ 0.05 versus isotonic condition. b Effect on RVD of the addition of verapamil (10-4 M) plus ATPcS (5 lM) to the

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time (min) hypotonic solution. The drugs were added 15 min prior to exposure to the hypotonic solution which also contained the drugs; n (number of cells) = 14. The values are means ± SE. * p \ 0.05 versus isotonic condition; ** p \ 0.05 versus maximum swelling

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Fish Physiol Biochem Glyben/ATPγS Hypotonic 1.35

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time (min) Fig. 9 Effect on RVD of the addition of glybenclamide (5 9 10-4 M) plus ATPcS (5 lM) to the hypotonic solution. The drugs were added 15 min prior to exposure to the hypotonic solution which also contained the drugs; n (number of cells) = 13. * p \ 0.05 versus isotonic condition

P2 purinergic receptors both in fish hepatocytes (Ollivier et al. 2006b; Pafundo et al. 2004) and in Necturus erythrocytes (Light et al. 2001). It is conceivable that the interaction with P2 receptors is required also in seabream hepatocytes since a recovery of cell volume was not observed when the hypotonic stress was applied in the presence of the P2 antagonist, suramin (Fig. 4). It is noteworthy that in the control conditions (Fig. 1), the cells exposed to the hypotonic solution swelled and maintained an enlarged size for about 20 min, after which time they underwent RVD, as already observed in our previous study (Faggio et al. 2011). In the presence of the P2 receptor agonist ATPcS, this time lag was strongly reduced. This could suggest that the delay in the onset of the homeostatic response observed in the control conditions is due to the time required to reach ATP extracellular concentrations sufficient to activate the P2 receptors and hence the RVD response, considering that the extracellular ATP concentration at any time depends on the ATP released from the cell and on the depletion by extracellular diffusion and by the activity of extracellular lytic enzymes (Chara et al. 2010). It is conceivable that ATPcS increases the concentration of extracellular ATP by a positive feedback mechanism, mimicking the ATP induced ATP release already proposed (Anderson et al. 2004; Chara et al. 2010; Locovei et al. 2006). In addition, it can be argued that ATPcS, which is relatively resistant to enzymatic degradation, overcomes the inhibitory effect on RVD

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produced by ATP metabolites, such as adenosine, acting on P1 receptors, as already shown by Pafundo et al. (2004). This conclusion is suggested by the results obtained with agonist and antagonist of P1 receptors. In fact, we observed that in the presence of the P1 antagonist 8PT, the RVD response did not show the delay observed in the control condition (Fig. 1), in addition the cells exhibited a complete recovery of cell volume (Fig. 5) that was not observed in the control cells (Fig. 1). On the other hand in the presence of the P1 agonist adenosine, the homeostatic response was impaired (Fig. 6). It is known that ATP can be released from the cells, beside by lysis, through regulated mechanisms such as conductive release through a channel, an efflux through a solute transporter and an exocytosis of ATP-enriched vesicles and/or an exocytosis of ATP and insertion of channels (Cantiello 2001; Chara et al. 2011; Fitz 2007; Praetorious and Leipziger Praetorius and Leipziger 2009; Roman et al. 2001). Even if we did not measure the release of ATP from the cells, we suggested that this mechanism exists also in seabream hepatocytes, based on the following observations: (1)

(2)

the lack of RVD in the presence of the ATP scavenger apyrase (Fig. 2) suggests that RVD depends on extracellular ATP. Since the control cells exhibit RVD (Fig. 1) and we did not add exogenous ATP in the incubation medium, we have to suppose that ATP is released from the cells. the observation that the P2 antagonist suramin completely inhibits RVD (Fig. 4) indicates that the activation of these receptors by ATP is necessary for RVD. It is logic to suppose that they are activated in the control experiments in which the cells exhibited RVD again, since exogenous ATP was not added to the incubation medium, we have to suppose that the activation is due by ATP released from the cells.

In an attempt to detect the mechanisms involved in ATP release from the cell in seabream hepatocytes, we performed some experiments with known inhibitors of the possible mechanisms of ATP release. The results we obtained suggest the involvement of the P-glycoprotein since the ATP analog, ATPcS, was able to counteract the inhibitory effect produced by verapamil, a P-glycoprotein inhibitor (Roman et al. 1997).


In fact, as shown in Fig. 7a, when the hypotonic shock was applied in the presence of verapamil, the isolated hepatocytes did not exhibit RVD, while they were able to recover cell volume when exposed to the hypotonic solution in the presence of verapamil plus ATPcS (Fig. 7b). In this respect, seabream hepatocytes would behave differently from those of turbot in which verapamil failed to affect ATP release following the hypotonic shock (Ollivier et al. 2006b). It is known that verapamil is an inhibitor of L-type Ca2? channels. Therefore, the possibility that ATPcS recovers the RVD inhibition resulting from calcium influx impediment cannot be excluded. However, if the effect of verapamil was only related to Ca2? influx inhibition, ATPcS would have had a stimulatory effect on RVD also in a calcium-free medium. On the other hand, this effect was not observed (Fig. 8). Our previous studies (Faggio et al. 2011) showed that glybenclamide was a potent inhibitor of regulatory volume decrease in seabream hepatocytes. We had suggested that the effect observed was due to an inhibition of swelling-activated Cl- channel, involved in the recovery of cell volume as previously suggested (Inoue et al. 2010; Liu et al. 1998), but we had not excluded the possibility that the drug acted on other mechanisms involved in RVD, such as the ATP release. This hypothesis was suggested by the results obtained by Ollivier et al. (2006b) showing that glybenclamide inhibited the ATP efflux from turbot hepatocytes and hence RVD by the participation of CFTR protein. Moreover, a role of this protein in the electrodiffusional ATP movement (Cantiello 2001) or in the stimulation of separate ATP channels (Braunstein et al. 2001) was also proposed. In seabream hepatocytes, the involvement of CFTR in the RVD response by a release of ATP seems unlikely because the ATP analog ATPcS was not able to counteract the inhibitory effect of glybenclamide on RVD (Fig. 9). However, other studies, by employing different experimental methods, are necessary to clarify the mechanism of ATP release from the cell. In conclusion, this videometric study would suggest that the swelling-activated ATP release from the cell is involved in the RVD response of isolated seabream hepatocytes. The nucleotide, probably released by a verapamil-sensitive mechanism, exerts its stimulatory effect on the homeostatic response by interaction with P2 receptors. On the other hand, the activation of P1 receptors by ATP metabolites produces opposite

effects, confirming similar observations on trout hepatocytes (Pafundo et al. 2004).

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