Alcohol consumption has long been associated with cell damage. Many studies on ethanol toxicity have been conducted following middle or long term exposition to ethanol [18, 27–30]. However, attention on the early steps initiated by ethanol is currently lacking. Furthermore, although relevant studies have been performed on the effect of ethanol on the oxidative state of cellular components and/or apoptosis [7, 8, 16, 28], its effects on Ca2+ homeostasis have not received much attention.
To date, it is not clear whether the deleterious actions of ethanol on cellular physiology could result from the production of toxic derivatives from alcohol metabolism or from a direct action of alcohol on cellular structures, which would be morphologically and functionally altered [12, 16, 27, 30–33].
Recently we have shown that ethanol, leading to a delayed or reduced Ca2+ extrusion from the cytosol towards the extracellular space or into the cytosolic stores, induces a cytosolic Ca2+ overload that could be the basis of alcoholic pancreatitis . However, the mechanisms by which ethanol exerted its effect was not clearly demonstrated, although it was postulated that ROS generation might be involved in the process.
In the present study, we have further investigated the possible mechanisms involved in the early effects of an acute ethanol exposure on CCK-8-evoked Ca2+ signals in mouse pancreatic acinar cells, because Ca2+ signalling is of critical importance for CCK-8-evoked responses in the exocrine pancreas.
Our results show that ethanol induces mobilization of Ca2+ in the form of oscillations, a pattern of Ca2+ mobilization that seems not to depend on the concentration of ethanol applied to the cells. To our knowledge, this is the first time to be shown that ethanol induces Ca2+ mobilization in pancreatic acinar cells at such concentrations, 1 to 50 mM, being the highest a concentration of ethanol found in plasma of intoxicated humans . In a former work, higher concentrations of ethanol were employed, up to 850 mM, and effects of lower concentrations were discarded . In addition, here we show that ethanol leads to a transformation of the typical oscillatory pattern of [Ca2+]i, evoked by a physiological concentration of CCK-8, into a sustained response usually evoked by supraphysiological concentrations of the agonist. Our results support previously reported works, which propose that ethanol-induced sensitization of the pancreatic acinar cell results in pancreatitis responses with low doses of CCK-8, that by itself does not cause pancreatitis . Our observations further support those shown by Petersen and Sutton , who propose that cell death results from excessive loss of Ca2+ from the endoplasmic reticulum, which is mediated by Ca2+ release through specific channels and inhibition of Ca2+ pumps in intracellular stores, followed by entry of extracellular Ca2+. All this together leads to abnormal global and sustained cytosolic Ca2+ signals.
In the present work, we have further studied the mechanisms by which ethanol evokes its deleterious effects in pancreatic acinar cells, in order to clarify whether the effects of ethanol are direct or mediated through its metabolization, and if ROS are somehow involved.
The inhibition of alcohol metabolization would increase those effects that are due to a direct action of ethanol, whereas it would block those effects of ethanol that would be due to its metabolites. Through our study, we have employed the ADH inhibitor 4-MP. This compound substantially decreased ethanol effects on connective tissue growth factor mRNA expression in mouse pancreatic stellate cells . Inhibition of ADH by 4-MP is concentration-dependent, with a range of action from 1 μM up to 1000 μM in different tissues [23, 24, 34]. Thus, the concentration of the ADH inhibitor we have employed falls within the concentration range successfully employed to inhibit ethanol metabolization by this enzyme.
Our results show that the effect of ethanol on the peak [Ca2+]i response evoked by CCK-8 was not blocked by the inhibition of ethanol metabolization by ADH; conversely, it was slightly increased. However, the "steady-state level" and the rate of decay of [Ca2+]i to basal values after CCK-8 stimulation in the presence of ethanol were significantly changed, i.e., [Ca2+]i recovered faster in the presence of ADH inhibition.
Our results could be explained on the basis of a direct action of ethanol onto the Ca2+-releasing mechanisms activated by CCK-8, and by an indirect action, mediated through ethanol metabolites, on the [Ca2+]i recovery mechanisms. We think that ethanol itself sensitizes the pancreas to the effects of other stimuli such as the physiological agonist CCK-8, acting on CCK-8-receptor or on the Ca2+ releasing channels. This could initially have an effect on the Ca2+ releasing mechanisms, and therefore would result in a bigger Ca2+ response of the tissue. This is reflected as a transformation of the physiological oscillations in [Ca2+]i into a supraphysiological sustained change in [Ca2+]i, and by the higher peak of [Ca2+]i observed in the presence of ethanol.
On the other hand, the recovery of [Ca2+]i is mainly carried out by the sarco-endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase, the pumps that actively transport Ca2+ into the endoplasmic reticulum and towards the extracellular medium respectively. A delay or decreased extrusion of Ca2+ from the cytosol will lead to an increased mobilization of Ca2+ compared to normal conditions. This can potentially lead to a cytosolic Ca2+ overload. Thus, t decrease in the generation of ethanol metabolites by inhibition of its metabolization would allow the Ca2+ transport mechanisms to effectively recover resting [Ca2+]i, as we have shown in the present study.
This is in agreement with our previous findings, which show that ethanol slows down Ca2+ transport into the ER and through the plasma membrane, which potentially leads to a cytosolic Ca2+ overload . However, the exact mechanisms by which ethanol evokes these effects need to be further clarified.
The antioxidant activity of proanthocyanidins is stronger than vitamin C or vitamin E in aqueous systems and its protective effects on diseases related to ROS have been demonstrated in a number of cell types [35–37]. Our results show that ethanol induces ROS production in mouse pancreatic acinar cells, in agreement with previous studies . Moreover, preincubation of pancreatic acinar cells in the presence of cinnamtanninB-1 significantly inhibited ethanol-stimulated ROS generation. This supports the hypothesis that ethanol metabolization could lead to ROS generation which, in turn, would affect Ca2+ transport mechanisms, and is in agreement with our previous findings [20, 21].
On the other side, preincubation of pancreatic acinar cells in the presence of this antioxidant did not block ethanol-induced increase in the peak of [Ca2+]i in response to CCK-8. This further supports the hypothesis that ethanol might be having direct effects on the Ca2+-releasing mechanisms in response to CCK-8, as suggested above. However, in the presence of the antioxidant, the "steady-state level" achieved after stimulation of cells with the hormone was lowered compared to that observed in the presence of ethanol, and the rate of decay of [Ca2+]i to basal values after CCK-8 stimulation was faster than in the presence of ethanol, reaching values similar to those observed after stimulation of cells with CCK-8 alone. This supports the hypothesis that ethanol metabolization could lead to ROS generation which, in turn, would affect Ca2+ transport mechanisms.
Therefore, here we can hypothesize that ethanol influences CCK-8-evoked changes in [Ca2+]i both by a direct and an indirect action, the latter mediated through ROS generation. Our findings are in agreement with others which show that ethanol can induce several cellular reactions which result in a modification of cellular red-ox status, leading to overproduction of ROS [16, 19, 38]. Nevertheless, involvement of non-oxidative metabolites from ethanol cannot be excluded.
In summary, our findings show that ethanol has dual effects on the physiology of intracellular Ca2+ signalling. First, ethanol induces a direct effect on the Ca2+ release mechanisms in response to CCK-8; and second, ethanol presents an indirect effect that is mediated, at least in part, by ROS generation following its metabolization. All this together leads to higher levels of [Ca2+]i following stimulation of cells with CCK-8. Ethanol will consequently lead to Ca2+ accumulation within the cytosol, creating a situation potentially leading to cytosolic Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.