THE ROLE OF INTRACELLULAR ORGANELLES IN Ca2+-HOMEOSTASIS IN SECRETORY CELLS OF THE LACRIMAL GLAND

It is known that different Ca2+-transport systems make distinct contribution to the formation of Ca2+-signal in various secretory cells. In recent years there has been a significant increase in interest in study of the lacrimal glands functioning. However, the results of study of the Ca2+-signalling in lacrimal gland have not yet been summarized in the literature. This review is devoted to analysis of functioning of Ca2+-transport system of secretory cells in the rat exorbital lacrimal gland. IP3Rs in exorbital lacrimal gland cells were effectively inhibited by 2-APB (10 μM) and activated by IP3 (2 μM), as well as by cholinomimetics, carbacholine (10 μM) and purine receptor agonists, ATP (1 mM). Signaling pathways activated by P2Y-receptors in the lacrimal gland secretory cells were partially mediated by IP3R activation. RyRs of lacrimal gland secretory cells were activated by Ca2+ and low concentrations of ryanodine (0,05–1 μM). Simultaneous activation of RyRs and IP3Rs in these cells caused Ca2+ release from the same store. Ca2+ mobilization from the intracellular stores induced by carbacholine (10 μM) or thapsigargin (1 μM) caused store-operated Ca2+ entry in secretory cells of the studied glands, which was partially inhibited by 2-APB. SERCA of exorbital lacrimal gland cells was efficiently inhibited by eosin Y (5–10 μM) and by thapsigrgin (1 μM). In contrast to permeabilized cells, the Ca2+ content in intact cells did not change under the influence of eosin Y (5–20 μM), indicating the inhibition of PMCA. Ca2+-ATPase activity of permeabilized cells of studied glands depended on the incubation time, the substrate amount and Ca2+ concentration in the incubation medium. Mitochondrial Ca2+-uniporter of lacrimal gland secretory cells was inhibited by ruthenium red (10 μM). Effects of ryanodine and ruthenium red on the Ca2+ content in cells were statistically significantly non-additive. Furthermore, ryanodine at concentrations of 1–3 μM caused a dose-dependent decrease in the respiration rate of the studied cells and this effect persisted after cells preincubation with ruthenium red or thapsigargin. This suggests that in addition to the endoplasmic reticulum RyRs activation, ryanodine inhibited the Ca2+ transport to the mitochondrial matrix, which was insensitive to the ruthenium red.

It is generally accepted that Ca 2+ plays a significant role in secretory process by the exocrine glands [2,13,19,40,41,57]. Ca 2+ concentration in cytosol is determined by two oppositely directed Ca 2+ flows. The inward flow (relative to cytosol) results from passive transport of Ca 2+ from the extracellular space through the plasma membrane and/ or Ca 2+ release from intracellular stores. Opposite in direction outward flow causes the reduce of cytosolic Ca 2+ due to its active output to the extracellular fluid or into intracellular stores [41].
It is known that different Ca 2+ -transport systems make distinct contribution to the formation of Ca 2+ -signal in various secretory cells. In recent years there has been a significant increase in interest in study of the lacrimal glands functioning. However, the results of study of the Ca 2+ -signalling in lacrimal gland have not yet been summarized in the literature. Ca 2+ -signaling in secretory cells of these glands is far less studied than in other types of glands. The number of publications that have at least indirect relation to Ca 2+ -transport systems of lacrimal glands is 2-4 or ders of magnitude less than the corresponding number of articles about organization of the Ca 2+ -signaling in other secretory glands. In the literature there are only few data regarding the identification and properties of some Ca 2+ -transport systems in the lacrimal gland secretory cells [5, 14, 21, 22, 25, 31, 34-38, 45, 60], while there is no syste matic analysis of their functioning, features and the role in Ca 2+ -signaling.
Understanding the mechanism involved in Ca 2+ -signalling in lacrimal gland secretory cells is essential for understanding of the regulation of their secretory process. These results can be the basis for the development of means for pharmacological correction of the dry eye syndrome, since both fluid and protein secretion by cells of lacrimal glands are Ca 2+ -dependent processes.
In our first article, published in Studia Biologica [35], we addessed the identification and features of SERCA and PMCA. The purpose of this review is to analyze the contribution of other intracellular Ca 2+ -transport systems in the Ca 2+ -homeostasis of the lacrimal gland secretory cells.
In the lacrimal gland secretory cells IP 3 Rs retain their functional activity in the dygitonin-permeabilized cells. The reduction of stored Ca 2+ under the influence of IP 3 is the direct evidence for this. In the presence of 2-APB (10 µM) in medium, statistically significant changes under the influence of IP 3 were not recorded ( Fig. 1) [36]. Despite low specificity of 2-APB, it is a clear proof of IP 3 Rs presence in the secretory cells of rat exorbital lacrimal gland.
The mechanism of signal transduction of P2Y receptors in the studied cells involves IP 3 R activation, since in presence of their inhibitor -2APB (10 µM) in the nominal Ca 2+free medium (that prevents P2X receptors activation), ATP-induced changes of Ca 2+ content were less expressive. This reduction of Ca 2+ in cells is really caused by the P2Y-receptors activation, since it was inhibited (but not completely) by suramin (100 µM) [36].
The mechanism of signal transduction in studied cells in conditions of both P2Y receptors and M-cholinergic receptors activation, at least partially, involved IP 3 R activation, but there were no data with which neurotransmitter (acetylcholine or noradrenaline) the ATP is released from nerve endings surrounding the lacrimal gland. When both M-cholinergic receptors and P2Y receptors were activated, Ca 2+ was released from the same IP 3 -sensitive store, since the effects of ATP and carbacholine at high concentrations (1 mM and 10 µM, respectively) on the Ca 2+ content were non-additive [36]. This is important in view of the physiological appropriateness clarification why various agonists activate Ca 2+ release from the same IP 3 -sensitive store, through the activation of common path of signal transduction.
It was also found [22] that stimulation of the lacrimal gland cells by acetylcholine caused the Ca 2+ -response with maximum amplitude, regardless of the time after stimulation by ATP. The authors concluded that ATP causes the release of only a small part of the available Ca 2+ , allowing acetylcholine to cause the re-release of Ca 2+ from the same store [22].
The release of stored Ca 2+ from the endoplasmic reticulum during the cell stimulation by agonists is due to the activation of IP 3 Rs and is amplified by RyRs activation. On basis of the results obtained by Western blot analysis and immunohistochemical methods it was found that all isoforms of RyRs are expressed in the lacrimal glands secretory cells of mice [45].
The dependence of RyRs sensitivity on Ca 2+ concentration is inherent for many types of cells [6,9,11,17,26,39,48]. This property was confirmed for the lacrimal gland secretory cells. Ryanodine (0,05-1 µM) reduces Ca 2+ content in the secretory cells of lacrimal glands in dose-dependent manner [32] (Fig. 2). On basis of the two-factor analysis of variance it was revealed that Ca 2+ concentration in the medium determined the Ca 2+ content in the investigated cells by 8.6 %, and the concentration of ryanodineby 87.9 %. The unaccounted factor amounted to only 3.49 % [32].  Simultaneous activation of RyRs and IP 3 Rs in these cells caused Ca 2+ release from the same store, since the effects of IP 3 (2 µM) and ryanodine (100 nM) on the total Ca 2+content in permeabilized secretory cells were statistically significantly non-additive [37].
Agonist-induced Ca 2+ release from the intracellular stores into the cytosol (and its outflow from cells) tends to reduce the pool of stored Ca 2+ [49], which in turn activates the store-operated Ca 2+ entry (SOCE) from the extracellular space [10,18,36,38].
It was shown that the store-operated calcium channels in the lacrimal gland secretory cells of mouse are formed by Orai1 [60]. Lacrimal acinar cells lacking Orai1 have diminished lacrimal fluid secretion following activation of the muscarinic receptor. These results also demonstrate the central role of SOCE in lacrimal exocrine function, since in Orai1 knockout mice calcium-dependent peroxidase secretion was eliminated [60]. Ca 2+ which entered the cell by SOCE is transported to the endoplasmic reticulum. The only system that provides Ca 2+ transport from the cytosol to the endoplasmic reticulum is SERCA (Ca 2+ , Mg 2+ -ATPase of endoplasmic reticulum) [41]. Primarily, thapsigargin [54] and eosin Y [30,35,53] belong to the inhibitors of Ca 2+ -pumps. It was shown that 2-APB is also capable to inhibit SERCA of the lacrimal gland secretory cells [31].
It should be noted that the results obtained by the ATPase activity measurement of permeabilized cell under the influence of these inhibitors [34] are in good agreement with the results obtained from measuring changes of the Ca 2+ content in cells [35]. Reduction of Ca 2+ content in permeabilized cells by thapsigargin (1 µM) -specific SERCA inhibitor was the same as by eosin Y (5 mM). In contrast to permeabilized cells, the Ca 2+ content in intact cells did not change under the influence of eosin Y (5-20 µM), indicating the PMCA inhibition [35].
The contribution of mitochondria to Ca 2+ -homeostasis in secretory cells. Mitochondria make a comprehensive impact on the regulation of Ca 2+ -signals in the secretory cells of various glands [8,50,61]. Ca 2+ uptake by mitochondria is realized particularly through Ca 2+ -uniporter in their inner membrane [29]. For its inhibition ruthenium red is often used [20,28]. Today the functional activity of mitochondrial Ca 2+ -uniporter in the lacrimal gland secretory cells is confirmed [4] and its interaction with other Ca 2+ -transport systems in cells was studied on basis of the estimation of additivity of the effects of appropriate inhibitors [37].
Ruthenium red (10 µM) -an inhibitor of Ca 2+ -uniporter and thapsigargin (1 µM)an inhibitor of SERCA reduced the Ca 2+ content in intact and permeabilized cells of lacrimal glands. The total effect of ruthenium red and thapsigargin on Ca 2+ content in cells was additive [37]. This suggests on the influence of these two inhibitors Ca 2+ is passively released from different stores. Similarly, effects of IP 3 (2 µM) and ruthenium red in permeabilized cells, and M-cholinergic receptors agonist carbacholine (10 µM) and ruthenium red on intact cells were additive [37]. Beside this, adding carbacholine to the polarographic chamber caused an intensification of respiration of the studied cells. Mitochondrial Ca 2+ -uniporter inhibition, which prevents the Ca 2+ flow in the matrix caused the levelling of carbacholine-stimulating effect, and IP 3 R inhibition partially eliminated the effect of carbacholine [33]. However, the effects of ryanodine (0.1 µM) and ruthenium red on total Ca 2+ -content in the lacrimal cells were non-additive (Fig. 5) [37].
A lack of ryanodine effect on a background of red ruthenium can be caused by several factors. First, ruthenium red may inhibit the RyRs of endoplasmic reticulum, which was previously demonstrated, in particular, for non-excitable cells [47]. But the described effects of ruthenium red on Ca 2+ content in the tissue of lacrimal gland, in our opinion, is not related to the RyRs inhibition, as the decrease of functional activity of these channels must be accompanied by an increase (rather than decrease) in Ca 2+ content in the tissue. The effect of ryanodine is associated not only with the Ca 2+ release from endoplasmic reticulum, but also with activation of mitochondrial RyRs (mRyRs), which were identified, for example, in the inner mitochondrial membrane of cardiomyocytes and were inhibited by ruthenium red [3].
Furthermore, ryanodine at concentrations of 1-3 µM caused a dose-dependent decrease in the respiration rate of the studied cells (Fig. 6) and this effect persisted after cells preincubation with ruthenium red or thapsigargin. This suggested that in addition to the endoplasmic reticulum RyR activation, ryanodine inhibited the Ca 2+ transport to the mitochondrial matrix, that was insensitive to the ruthenium red [37].

SUMMARY
Based on our results and literature data, we suggested the following scheme of Ca 2+ -transport systems functioning in lacrimal gland secretory cells (Fig. 7).  Primary mediators for lacrimal gland secretory cells are ATP -the agonist of the P2X and P2Y-purinoreceptors and acetylcholine and its analogues -agonists of the identified M 3 -cholinergic receptors [12,22,23,36,43].
Signalling pathways activated by the P2Y-receptors in the lacrimal gland secretory cells were partially mediated by the IP 3 R activation [36]. IP 3 Rs of exorbital lacrimal gland cells were effectively inhibited by 2-APB (10 µM) [36], which was previously demonstrated for other cell types [42,46,56,58,59]. When both M-cholinergic receptors and P2Y receptors were activated, Ca 2+ was released from the same IP 3 -sensitive store since the effects of ATP and carbacholine at high concentrations (1 mM and 10 µM, respectively) on the Ca 2+ content were non-additive [36].
An increase in cytosolic Ca 2+ concentration after the IP 3 R and RyR activation is accompanied by activation of SERCA, PMCA, Ca 2+ -uniporter and mRyRs.
In addition to RyR activation in the endoplasmic reticulum, ryanodine inhibited the Ca 2+ transport to the mitochondrial matrix, that was insensitive to the ruthenium red [37]. This effect can be explained by presence of RyRs in the mitochondrial membrane (previously demonstrated to cardiomyocytes [3]), that may be important for regulating of cell energy supply.
Adding carbacholine to polarographic chamber caused an intensification of respiration of studied cells [33]. These results are in good agreement with those obtained previously by another group [24]. This effect is associated with Ca 2+ intake in the mitochondrial matrix and subsequent activation of the mitochondrial oxidation. Mitochondrial Ca 2+ -uniporter inhibition, which prevents the Ca 2+ flow in the matrix caused the levelling of carbacholine-stimulating effect, and IP 3 Rs inhibition partially eliminated the effect of carbacholine. It has an important general biological significance because it allows the regulation of mitochondrial respiration by a direct positive feedback, long before there would be a lack of ATP and triggers the inverse regulatory effects, homeostatic (not signaling) by their nature.
Further studies of the Ca 2+ -transport system functioning in the lacrimal gland secretory cells addesses of their functioning changes during aging and regulation by hormones.