Rat Lacrimal Gland Cells Research Articles

<b>α1-Adrenoceptors relate Ca</b><sup><b>2+</b></sup><b> modulation and protein secretions in rat lacrimal </b><b>gland </b>

Noradrenaline (NA) is a catecholamine with multiple roles including as a hormone and a neurotransmitter. Cellular secretory activities are enhanced by adrenergic stimuli as well as by cholinergic stimuli. The present study aimed to determine which adrenoceptors play a role in controlling intracellular calcium ion ([Ca(2+)]i) level in acinar cells of rat lacrimal glands. Expression of mRNA for adrenoceptor subtypes in the acinar cells was assessed using RT-PCR. All types except α2c, β1, and β3 were detected. NA induced a [Ca(2+)]i increase with a biphasic pattern in the acinar cells. Removal of extracellular Ca(2+) and use of Ca(2+)-channel blockers did not inhibit the NA-induced [Ca(2+)]i increases. In contrast, U73122 and suramin almost blocked these increases. The α1-adrenoceptor agonist phenylephrine induced a strong increase in [Ca(2+)]i. However, clonidine and isoproterenol failed to induce a [Ca(2+)]i increase. The peroxidase activity was quantified as a measure of mucin secretion. Ca(2+)-dependent exocytotic secretion of peroxidase was detected in rat lacrimal glands. The RT-PCR results showed that MUC1, MUC4, MUC5AC, MUC5B, and MUC16 were expressed in acinar cells. These findings indicated that NA activates α1-adrenoceptors, which were found to be the main receptors in Ca(2+)-related cell homeostasis and protein (including mucin) secretion in lacrimal glands.

Ca2+-транспортувальні системи секреторних клітин зовнішньоорбітальної сльозової залози щура. I. Ca2+-помпи плазматичної мембрани та ендоплазматичного ретикулуму

The mechanism of secretion by lacrimal glands, the innervation and orientation effects, and the mechanism of transduction of primary agonists are similar to the major salivary glands. However, unlike the major salivary glands, about organization of Ca 2+ signalling and Ca 2+ -transporting systems contribution to Ca 2+ homeostasis maintenance in secretory cells of the lacrimal glands is not totally clear yet. The purpose of the work was to investigate the role of plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca 2+ pumps in maintenance of Ca 2+ homeostasis in the secretory cells of the lacrimal glands. The study was performed on intact and digitonin-permeabilized secretory cells of exorbital lacrimal glands of rats. The functioning of the Ca 2+ -transporting systems was estimated by changes of Ca 2+ content in cells, after incubation with agonists or antagonists. Ca 2+ content was determined using metalochromic dye Arsenazo III. The baseline Ca 2+ content in intact and permeabilized cells decreases during incubation time. This decrease is caused by dysfunction of SERCA. This pump is high effectively inhibited by eosin Y (10 µmol/l) and thapsigargin (1 µmol/l). PMCA plays important role in maintaining of Ca 2+ homeostasis in the lacrimal gland secretory cells. Thus, the content of Ca 2+ in intact cells of rat lacrimal glands did not change under inhibition by eosin Y in wide concentration range (5–20 µmol/l) (unlike digitonin-permeabilized cells). In addition, eosin Y (5 µmol/l) prevented time-dependent reduction of Ca 2+ content in intact cells. In both cases, inhibition of PMCA eliminates the effect of inhibition of SERCA. Results of two-factor analysis of variance confirmed that in the absence of eosin Y incubation time and permeabilization of plasma membrane significantly affect the content of Ca 2+ in cells. At the same time, in the presence of eosin Y (5 µmol/l) only permeabilization of plasma membrane reliably affects of the changes of Ca 2+ content. Because in permeabilized cells, as well as eosin Y influence in concentration 5 µmol/l, PMCA does not transport Ca 2+ from the cell. Keywords: eosin Y, Са 2+ transport systems, Са 2+ pump of plasma membrane (PMCA), Са 2+ pump of endoplasmic reticulum (SERCA), lacrimal gland, secretory cells.

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5'-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺](i)) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺](i) in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺](i) increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺](i) increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺](i), whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺](i). A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺](i), although UTP (a P2Y₂,₄,₆ receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP >>> UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺](i), but α,β-meATP (a P2X₁,₃ receptor agonist) had no effect. RT-PCR indicated that P2X₂,₃,₄,₅,₆,₇ and P2Y₁,₂,₄,₁₂,₁₄ are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.

Cholinergic Agonists Activate P2X7Receptors to Stimulate Protein Secretion by the Rat Lacrimal Gland

To determine the interaction of M3 muscarinic receptors (M3AChR) and P2X(7) receptors to increase intracellular [Ca2+] ([Ca2+]i) and stimulate protein secretion in rat lacrimal gland cells. Exorbital lacrimal glands from male Sprague-Dawley rats were divided into pieces or digested with collagenase to form acinar clumps. [Ca2+]i was measured using an imaging system in acini incubated with fura-2/AM. Adenosine triphosphate (ATP) release was determined using the luciferin-luciferase reaction. Peroxidase secretion, our index for protein secretion, was measured spectrophotometrically. Acini were stimulated with the P2X7 receptor agonist, (benzoylbenzoyl)adenosine 5' triphosphate (BzATP), cholinergic agonist carbachol, or the activator of conventional and novel PKC isoforms, phorbol 12-myristate 13-acetate (PMA). The increase in [Ca2+]i caused by carbachol and BzATP used simultaneously was less than additive, but the increase in protein secretion was additive. The M3AChR antagonist atropine blocked the BzATP-stimulated increase in [Ca2+]i and peroxidase secretion. The P2X7 antagonist did not alter the carbachol-stimulated increase in [Ca2+]i or peroxidase. PMA- and BzATP-stimulated increases in [Ca2+]i were additive. Neither constitutively active PKCα, dominant-negative PKCα, nor PKCε altered BzATP-stimulated increases in [Ca2+]i. Carbachol increased ATP release from lacrimal gland pieces but not from acini. In lacrimal gland cells, the activation of M3AChRs stimulates P2X7 receptors to increase [Ca2+]i and protein secretion. The underlying mechanisms are unknown but could include the release of ATP or intracellular interactions not mediated by PKC isoforms. In addition, M3AChRs use signaling pathways that overlap with those used by P2X7 receptors to increase [Ca2+]i, but they also use signaling pathways not used by P2X7 receptors to stimulate protein secretion.

Expression of volume-sensitive Cl(-) channels and ClC-3 in acinar cells isolated from the rat lacrimal gland and submandibular salivary gland.

1. The expression of ClC-3 was examined in rat lacrimal gland and submandibular salivary gland cells using RT-PCR and Western analysis. Whole-cell patch clamp methods were used to investigate the expression of volume-sensitive anion channels in acinar cells isolated from these tissues. 2. Expression of mRNA encoding ClC-3, and ClC-3 protein, was found in rat submandibular gland by RT-PCR and Western analysis. Rat lacrimal gland cells, however, did not appear to express mRNA encoding for ClC-3, nor the ClC-3 protein. 3. Volume-sensitive anion conductances were observed in both rat lacrimal gland and submandibular salivary gland acinar cells. The conductance was of a similar size in the two cell types, but was much slower to activate in the lacrimal cells. 4. The properties of the conductances in lacrimal and submandibular cells were similar, e.g. halide selectivity sequence (P(I) > P(Cl) > P(aspartate)) and inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoate and tamoxifen. 5. The data suggest that the expression of ClC-3 is not an absolute requirement for the activity of volume-sensitive anion channels in rat lacrimal gland acinar cells.

High density of Ca(2+)-dependent K+ and Cl- channels on the luminal membrane of lacrimal acinar cells.

Tight-seal whole-cell recording and Ca2+ imaging were simultaneously performed on cell clusters or individual acinar cells of rat lacrimal glands during application of the secretagogue acetylcholine. Activation of Ca(2+)-dependent K+ and Cl- currents was selectively followed as a function of time by placing the cell potential near the equilibrium potential for Cl- or for K+ ion, respectively. Upon acetylcholine application to cell clusters, K(+)- and Cl(-)-selective currents displayed a distinctive initial rise ("hump"). At this time, there was only a small elevation of Ca2+ concentration, [Ca2+]i, that was restricted to the luminal end of acinar cells. A quantitative analysis of Ca2+ and current signals during the hump suggested that the luminal membrane contained high densities of K(+)- and Cl(-)-selective channels, roughly 10 times higher than those found in the basolateral domain. Distinct luminal and basolateral membrane domains were preserved in isolated cells, but with less contrasted densities than in cell clusters. The results suggest that Ca(2+)-dependent K+ channels are implicated not only in the transfer of salt from the blood compartment to the interior of acinar cells, as commonly accepted, but also in the electrolyte secretion from the cell interior to the acinar lumen.

Protein kinase C‐mediated desensitization of the muscarinic response in rat lacrimal gland cells.

1. Desensitization of the Ca2+ release response evoked by acetylcholine in acinar cells from rat lacrimal glands was studied using the Ca(2+)-sensitive dye Fura-2. The evolution of the amplitude and half-maximal rise time (t1/2) of the Ca2+ response was followed as a function of trial number in a series of stimulations. 2. Under control conditions repetitive applications of acetylcholine (15 s long applications every minute) led to a linear decrease of the amplitude and to a linear increase of t1/2 with trial number. Both amplitude and t1/2 recovered their control values after 20 min of washing. 3. Staurosporine (0.2-1 microM), an inhibitor of protein kinase C, was found to decrease the slopes of the variation of amplitude and t1/2 with trial number. 4. Prolonged treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C (100-250 nM for 2-4 h), also led to a markedly decreased desensitization, presumably as a result of down-regulation of protein kinase C. On the other hand moderate pre-treatments with TPA (16-32 nM for 10 min) strongly inhibited the response, most probably as a result of protein kinase C activation. 5. Application of oleoylacetylglycerol (50 microM), a weaker activator of protein kinase C, inhibited the response and enhanced desensitization. These effects were, however, not obtained after down-regulation of protein kinase C with strong exposure to TPA. 6. We conclude that protein kinase C activation following the ACh-induced Ca2+ rise and the concomitant diacylglycerol production mediates desensitization of the response. 7. Arachidonic acid (100 microM) inhibited the ACh-induced response and enhanced desensitization. However, this effect did not appear to be mediated by protein kinase C since it was also obtained with docosahexaenoic acid, an analogue of arachidonic acid which does not activate protein kinase C.

Measurement of Na-K pump current in acinar cells of rat lacrimal glands

Isolated cells from rat lacrimal glands were voltage clamped using the tight-seal whole-cell recording technique. The intracellular solution contained ATP and an elevated Na concentration (70 mM). Removing external K ions elicited an inward current shift. Ouabain (0.5 mM) induced an inward current shift of identical amplitude, but with slower kinetics. In the presence of ouabain, removal of K ions did not alter the cell current. The potassium- and ouabain-sensitive current was outward between -120 and +20 mV, and its amplitude decreased below -60 mV. This current was highly sensitive to temperature, and was not affected by blockers of the K channels which are present in these cells. It was attributed to an inhibition of the Na-K pump. The Na-K pump current was estimated to be 15 pA for an average acinar cell at physiological temperature, with 70 mM internal Na ions and 20 mM external K ions. Implications of this value in terms of electrolyte secretion are discussed.

Acetylcholine-induced closure of gap junction channels in rat lacrimal glands is probably mediated by protein kinase C.

In rat lacrimal gland cells, application of acetylcholine (ACh) opens Ca-dependent channels and closes gap junction channels. We have shown previously that the increase in intracellular calcium concentration induced by ACh, is not required for the closure of gap junctions. We have examined the effects of activators of protein kinase C on gap junction conductance measured with the double patch-clamp technique. This conductance was markedly reduced by incubating the cell pairs for a few minutes with 100 nM phorbol dibutyrate (PdBu). Two membrane permeant analogues of diacylglycerol, OAG (1-oleoyl-2-acetyl-glycerol) and DOG (1,2-dioleoyl-glycerol) also induced a closure of gap junction channels. This effect was observed in the concentration range 10-100 microM when the diacylglycerol was added on intact cells, and at 75 microM when it was applied on dialysed cells. The cell uncoupling was not mediated by phosphatidate, a degradation product of OAG, nor by a phospholipase A2-induced increase in arachidonate concentration. The OAG-induced closure of gap junction channels reversed spontaneously upon prolonged exposure (more than 90 min at 37 degrees C) to 25 microM OAG. After a prolonged OAG treatment, the ability of ACh to uncouple the cells was markedly reduced. ACh induced uncoupling was modulated to some extent by intracellular Ca and had an absolute requirement for Mg. These results indicate that ACh-induced closure of gap junction channels may be mediated by PKC.

Muscarinic activation of ionic currents measured by a new whole-cell recording method.

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].

Potentiation of muscarinic and alpha-adrenergic responses by an analogue of guanosine 5'-triphosphate.

Ca2+-dependent K+ and Cl- currents were recorded in isolated and dialyzed rat lacrimal gland cells by use of the tight-seal whole-cell recording technique. Under control conditions, application of acetylcholine (0.5-1.0 microM) resulted in the full activation of both types of current. When 50-200 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S], a nonhydrolyzable GTP analogue) was added to the intracellular solution, activation of both currents was seen with 1 nM acetylcholine, a dose 1/100th that needed under control conditions. Dialysis with solutions containing 200 microM GTP or cAMP had no, or only slight, potentiation effects. The effects of GTP[S] were obtained only when ATP was included in the intracellular solution. The potentiated responses to acetylcholine were blocked by increasing 10-fold the intracellular Ca2+-buffering capacity and were not dependent on external Ca2+. Thus, the potentiated responses appeared to result from a release of Ca2+ from internal stores. GTP[S] also greatly potentiated the Ca2+-dependent adrenergic (norepinephrine) response of this preparation. In addition, GTP[S] elicited in some cells transient responses without application of acetylcholine or norepinephrine. Finally, rapid and sustained responses were seen as soon as the cells were dialyzed with inositol trisphosphate (20 microM). These findings are discussed in terms of a possible role of a GTP-binding protein as a link between activation of muscarinic or adrenergic receptors and initiation of Ca2+ release by inositol trisphosphate.

Peroxidase Secretion from rat lacrimal gland cells in vitro: II. The role of calcium in stimulus-secretion coupling

The role of calcium in stimulus-secretion coupling in rat lacrimocytes was investigated. Monolayers of lacrimocytes released peroxidase into the incubation medium upon stimulation with calcium ionophore A 23187, l-norepinephrine, l-phenylephrine and tyramine. Our lacrimocyte preparation can be considered free of endogenous catecholamines, because lacrimocytes derived both from normal and from reserpinized rats responded to tyramine with respect to peroxidase secretion. Phenoxybenzamine, cocaine, verapamil and sodiumorthovanadate failed to inhibit the l-phenylephrine-evoked peroxidase secretion. The stimulatory effects of A 23187, l-norepinephrine and l-phenylephrine required extracellular Ca 2+ and could be blocked by phentolamine. The action of tyramine was insensitive to all these measures. It is suggested that the stimulus-secretion of peroxidase from lacrimocytes may consist of a Ca 2+-dependent phase for stimulants as A 23187, l-norepinephrine and l-phenylephrine and a Ca 2+-independent phase for the non-specific tyramine action.

Peroxidase secretion from rat lacrimal gland cells in vitro I. Alpha-adrenergic stimulation in the absence of alpha-adrenoceptors

The identification of α-adrenergic receptors and subdivision into α 1- or α 2-subtypes were studied by measuring the specific binding of the radioligands [ 3H]-prazosin as well as [ 3H]-clonidine to membranes prepared from homogenized rat lacrimal glands. The absence of high-affinity binding for [ 3H]-prazosin as well as for [ 3H]-clonidine indicates that rat lacrimal glands do not possess a substantial amount of α 1- and α 2-adrenoceptors. The binding data correspond with the characterization by pharmacological means. Monolayers of lacrimocytes were incubated with various selective α 1-, α 2-, β-adrenergic agonists and antagonists or other substances, and peroxidase discharge was measured over a period of 1 hr. Among various substances only l-norepinephrine, l-phenylephrine, tyramine and ionophore A 23187 were stimulants of peroxidase secretion, whereas the adrenergic-stimulated secretory response was only suppressed by phentolamine.

Alpha-adrenoceptor control of peroxidase secretion from rat lacrimal gland cells in vitro

This study presents a modified technique for dissociating rat exorbital lacrimal gland tissue into single cells for the in vitro study of enzyme secretion. Lacrimal gland slices were treated with collagenase and hyaluronidase followed by slight mechanical agitation and rinsing in fresh medium. Scanning electron microscopy showed that stromal elements disappeared after enzyme digestion and that isolated lacrimocytes have surface blebs, microvilli and ruffles. Lacrimocytes monitored by transmission electron microscopy showed a loss of secretion granules upon α -adrenergic stimulation. Monolayers of lacrimocytes were incubated with various selective α 1 -, α 2 - and β -adrenergic agonists and antagonists, and peroxidase as well as β -hexosaminidase release was measured over a period of 60 min. It appeared that the secretion of peroxidase only is enhanced by stimulation of α 1 -adrenoceptors.