Interaction of α_1D-Adrenergic and P2X₇ Receptors in the Rat Lacrimal Gland and the Effect on Intracellular [Ca²⁺] and Protein Secretion

Abstract

Purpose.: To determine whether α_1D-adrenergic receptors (α_1D-AR) and P2X₇ receptors interact by determining their effect on ATP release, intracellular [Ca²⁺] ([Ca²⁺]_i), and protein secretion in rat lacrimal gland acini.

Methods.: Exorbital lacrimal glands from male Sprague-Dawley rats were divided into pieces or digested with collagenase to form acini. With the use of an imaging system, [Ca²⁺]_i was measured in acini loaded with fura-2. 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) or the α_1D-AR agonist phenylephrine with or without antagonist preincubation.

Results.: Phenylephrine increased ATP release from pieces in a time-dependent manner. The α_1D-AR antagonist BMY7378 blocked the BzATP-stimulated increase in [Ca²⁺]_i but not in peroxidase secretion. The P2X₇ antagonist A438079 blocked the phenylephrine-stimulated increase in [Ca²⁺]_i but not peroxidase secretion. The increase in [Ca²⁺]_i caused by phenylephrine and BzATP used simultaneously or sequentially was additive, as was the increase in peroxidase secretion. The inhibition of protein kinase C isoforms or calcium calmodulin kinase II did not alter the BzATP-induced increase in [Ca²⁺]_i.

Conclusions.: The authors conclude that activation of α_1D-AR releases ATP, which induces P2X₇ receptors to increase [Ca²⁺]_i but not to stimulate protein secretion. P2X₇ receptors in turn activate α_1D-AR to increase [Ca²⁺]_i but not to stimulate protein secretion. Furthermore, α_1D-AR compared with P2X₇ receptors use different cellular mechanisms to increase [Ca²⁺]_i and cause protein secretion.

The lacrimal gland secretes proteins, electrolytes, and water into the tear film and helps maintain the health of the cornea and conjunctiva. When the volume or composition of secreted lacrimal gland fluid changes, the structure and function of the cornea and conjunctiva are altered, and dry eye results. Thus, identifying the agonists that stimulate lacrimal gland secretion and the intracellular signaling pathways used by these agonists is critical in describing the normal regulation of secretion. This knowledge forms the basis for determining dysfunction caused by lacrimal gland pathology in dry eye. 

Nerves are the predominant stimuli of lacrimal gland secretion. ¹ The lacrimal gland is innervated by efferent sympathetic and parasympathetic nerves that release the neurotransmitters norepinephrine (from sympathetic nerves) and acetylcholine (Ach) and VIP (from parasympathetic nerves). Norepinephrine, acetylcholine, and VIP are each potent and effective stimuli of lacrimal gland secretion, especially protein secretion, and each activates a separate, distinct signaling pathway. ^{2 –5} Norepinephrine activates α_1D-adrenergic receptors (α_1D-AR), which cause an increase in [Ca²⁺]_i by a mechanism that is not yet determined but is not by production of inositol 1,3,5-trisphosphate (InsP₃). ⁴ In addition, these receptors activate endothelial nitric oxide synthase to produce NO. ⁶ The NO activates guanylyl cyclase to increase cellular levels of cGMP, which phosphorylates specific substrates to stimulate protein secretion. ⁶ Stimulation of α_1D-AR, also using an unknown effector enzyme, produce diacylglycerol, which activates protein kinase Cε (PKCε) to stimulate secretion and PKCα and PKCδ to inhibit secretion. ⁵ α_1D-AR also transactivate the epidermal (EGF) receptor to increase extracellular-regulated kinase (ERK)1/2 activity, which attenuates secretion. ^7,8 Acetylcholine activates muscarinic type 3 acetylcholine receptors (M₃AchRs), which are coupled to phospholipase Cβ (PLCβ). PLCβ activation produces the PKC activator diacylglycerol and InsP₃. ³ InsP₃ increases the [Ca²⁺]_i that, along with the activation of PKCα, -δ, and -ε, stimulates the secretion of protein stored in preformed secretory granules. ^3,5 M₃AchR also activate ERK 1/2 and phospholipase D, which attenuate secretion. ^9,10 VIP interacts with VIPAC1 to stimulate secretion by increasing cellular levels of cAMP and increasing [Ca²⁺]_i. ¹¹ Even though norepinephrine, Ach, and VIP activate distinct signaling pathways, the neurotransmitters can be released together and can interact, causing a different secretory response than that activated by each agonist alone. For example, phenylephrine and VIP added together potentiate secretion, ² whereas phenylephrine and carbachol (an Ach analog) added at the same time cause additive secretion. ⁴  

Most cell types can release ATP, which activates another type of receptor, purinergic receptors. P2 purinergic receptors are divided into two subtypes, P2Y and P2X. P2Y receptors are metabotropic, G protein–linked receptors that increase [Ca²⁺]_i by activating PLCβ to produce InsP₃, as does the M₃AchR in the lacrimal gland. P2X receptors are ionotropic and nonselective ion channels that increase [Ca²⁺]_i by inducing Ca²⁺ influx. In lacrimal gland acini, ATP predominantly activates P2X rather than P2Y receptors. Even though all P2X receptors except P2X₅ are present in the lacrimal gland, only P2X₃ and P2X₇ appear to be functional because they increase [Ca²⁺]_i and stimulate protein secretion. ¹²  

We recently examined the interaction of M₃AchR- and P2X₇-induced responses. We found that M₃AchR activates P2X₇ receptors by releasing ATP from cells other than acinar cells and by an intracellular interaction. ¹³ M₃AchR stimulation of P2X₇ receptors increases [Ca²⁺]_i and induces protein secretion. Compared with P2X₇ receptors, M₃AchR use additional cellular mechanisms to induce protein secretion. 

In the present study we investigated the interaction between α_1D-AR and P2X₇ receptors by determining their effect on ATP release, [Ca²⁺]_i, and protein secretion. We found that activation of α_1D-AR releases ATP from acini, which induces P2X₇ receptors to increase [Ca²⁺]_i but not to stimulate protein secretion. P2X₇ receptors, in turn, activate α_1D-AR to increase [Ca²⁺]_i but not to stimulate protein secretion. Furthermore, α_1D-AR and P2X₇ receptors use different cellular mechanisms to increase [Ca²⁺]_i and cause protein secretion. Interaction of P2X₇ receptors with α_1D-AR is different from the interaction of P2X₇ receptors with M₃AchR. 

Materials and Methods

Materials

Fura-2 tetra-Acetoxyl-methyl ester (fura-2/AM) and reagent (Amplex Red) were purchased from Invitrogen (Carlsbad, CA). Collagenase (CLSIII) was from Worthington Biochemicals (Lakewood, NJ), whereas CSG 9343B was from Tocris Bioscience (Ellisville, MO). All other chemicals were from Sigma-Aldrich (St. Louis, MO). 

Animals

All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Schepens Eye Research Institute Animal Care and Use Committee. Male Sprague-Dawley rats weighing 125 to 150 g were purchased from Taconic Farms (Germantown, NY). Rats were maintained in constant temperature rooms with fixed 12-hour light/12-hour dark intervals of 12 hours and were fed ad libitum. They were anesthetized for 1 minute in CO₂ and then decapitated. Both exorbital lacrimal glands were removed immediately. 

Preparation of Lacrimal Gland Acini

Collagenase digestion was used to prepare acini. Lacrimal glands were fragmented before incubation at 37°C with collagenase (100 U/mL, CLSIII; Worthington Biochemicals) in Krebs-Ringer bicarbonate buffer with HEPES (KRB-HEPES) (119 mM NaCl, 4.8 mM KCl, 1.0 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 10 mM HEPES, and 5.5 mM glucose, at pH 7.45) plus 0.5% BSA for 30 minutes. After incubation, fragments were triturated, filtered through a nylon mesh (150-μM pore size), and centrifuged at 50g for 2 minutes. The pellet was washed twice through KRB-HEPES containing 4% BSA. The dispersed acini recovered for 60 minutes at 37°C before use. 

Measurement of [Ca²⁺]_i

Acini were incubated for 30 minutes at room temperature in the dark with KRB-HEPES containing 0.5% BSA, 0.5 μM fura 2/AM, 8 μM pluronic acid F127, and 250 μM sulfinpyrazone followed by washing in sulfinpyrazone. Calcium measurements were made with a ratio imaging system (InCyt Im2; Intracellular Imaging, Cincinnati, OH) using excimer wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. At least 10 clumps of acini were used for each condition. Inhibitors or antagonists were added 30 minutes before agonists (Table 1). CSG 9343B and calphostin C were dissolved in dimethyl sulfoxide (DMSO; final concentration, 0.1%); all others were dissolved in dH₂O. After the addition of antagonists, inhibitors, and agonists, data were collected in real time. Data are presented as the actual [Ca²⁺]_i with time or as the change in peak [Ca²⁺]_i. Change in peak [Ca²⁺]_I was calculated by subtracting the average of the basal value (no added agonist) from the peak [Ca²⁺]_i. Although the data are not shown, the plateau [Ca²⁺]_i was affected similarly to the peak [Ca²⁺]_i. All [Ca²⁺]_i measurements in the presence of BzATP were performed in the absence of extracellular Mg²⁺ to increase the P2X₇ receptor Ca²⁺ response. 

Table 1.

 

Inhibitors and Their Target Molecules

Inhibitor	Target
BMY 7378	α1D-Adrenergic receptors
A438079	P2X7 purinergic receptors
Apyrase	ATPases
Calphostin C	Protein kinase C (PKC)
CGS 9343B	Ca2+/calmodulin-dependent kinase

Measurement of ATP Release

Lacrimal glands were removed and minced into small pieces. Pieces, or acini, were placed into cell strainers and preincubated in 0.5% BSA in KRB for 1 hour in a 12-well culture dish at 37°C. The strainers were moved to new wells containing fresh 0.5% BSA in KRB for an additional hour. Pieces were then placed in fresh 0.5% BSA in KRB containing 10⁻⁴ M ARL-67516, α,β methylene adenosine diphosphate, and β,γ methylene ATP, which are inhibitors of ectonucleotide pyrophosphatases and ectoATPases. Inhibitors were added 30 minutes before agonists (Table 1). Agonists were added for 0 to 10 minutes, and the lacrimal gland pieces were removed. The ATP concentrations in the supernatants were determined with the chemiluminescent luciferin-luciferase reaction using an ATP assay kit (FLAA; Sigma-Aldrich). The luciferin-luciferase working solution was 100 μL stock solution to 1 mL assay buffer, according to the manufacturer's instructions. Twenty-five microliters of samples were added to a 96-well plate and placed in a luminometer (SynergMx microplate reader; BioTek Instruments, Winooski, VT). The luciferin-luciferase working solution (15 μL) was injected into each well using the internal injector system. The emitted light was recorded with an integration time of 100 ms/measurement every minute for 10 minutes. ATP levels were calculated by integrating under the luminescence curve. The luminescence was converted to ATP concentrations using an ATP standard curve. Data were expressed as fold increase above basal. 

Measurement of Peroxidase Secretion

Acini were incubated for 40 minutes in KRB-HEPES containing 4% BSA at 37°C in the presence of agonists. Inhibitors or antagonists were added 30 minutes before agonists (Table 1). CSG 9343B and calphostin C were dissolved in DMSO (final concentration, 0.1%); all other compounds were dissolved in water. To terminate the incubation, acini were pelleted by centrifugation, and the supernatant was collected. The pellet was homogenized in 10 mM Tris-HCl (pH 7.5). Peroxidase activity, an index of protein secretion, was measured in duplicate in both the supernatant and the pellet. Peroxidase was measured using a reagent (Amplex Red; Invitrogen) that, when oxidized by peroxidase in the presence of hydrogen peroxide, produces a highly fluorescent molecule. The amount of fluorescence in the supernatant and pellet was quantified using a fluorescence microplate reader (model FL600; Bio-Tek Instruments) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Peroxidase was expressed as a percentage of peroxidase secreted into the media (supernatant) compared with the total peroxidase present in the cells before stimulation (pellet plus supernatant). Data were expressed as fold increase multiplied by basal, which was set to 1. 

Statistical Analysis

Results were expressed as mean ± SEM. Data were analyzed by Student's t-test. P < 0.5 was considered statistically significant. 

Results

α_1D-Adrenergic Agonists Increase [Ca²⁺]_i in a Concentration-Dependent Manner

When the [Ca²⁺]_i was determined in a large population of acini suspended in a cuvette, the α_1D-AR agonist phenylephrine increased [Ca²⁺]_i in a concentration-dependent manner. ⁴ A maximum increase of 15 nM was induced by phenylephrine at 10⁻³ M⁴. In the present experiments when measuring [Ca²⁺]_i by fluorescence microscopy in approximately 10 to 20 clumps of acini, phenylephrine also caused an increase in [Ca²⁺]_i (Fig. 1A). A maximum increase in [Ca²⁺]_i of 128.6 ± 17.8 nM was obtained with 10⁻⁵ M phenylephrine. Using the microscopy measurement technique, the change in [Ca²⁺]_i caused by supramaximal concentrations of phenylephrine caused a decreased response, unlike the cuvette method, with which the response remained elevated (Fig. 1B). 

Figure 1.

 

Effect of phenylephrine concentration on [Ca²⁺]_i in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing Fura-2 with increasing concentrations of the α_1D-adrenergic agonist phenylephrine (phenylephrine) added after 15 seconds of incubation. The trace shown (A) is the mean of four experiments. Mean change in peak [Ca²⁺]_i from four experiments (B). Values are mean ± SEM. Arrow: time of phenylephrine addition. *Significant difference from no addition (0).

α_1D-Adrenergic Agonists Release ATP from Lacrimal Gland Pieces

We sought to determine whether α_1D-AR agonists release ATP that could activate P2X₇ receptors. Pieces of lacrimal gland were stimulated with no agonists (basal) or the α_1D-adrenergic agonist phenylephrine (10⁻⁴ M) for 0 to 10 minutes. Basal ATP release was set to 1. Phenylephrine increased ATP release in a time-dependent manner with a significant increase in the release of ATP by 2.3 ± 0.04-fold at 0.5 minutes (Fig. 2A). Preincubation with BMY 7378, an α_1D-AR antagonist, completely blocked phenylephrine-stimulated ATP release (Fig. 2B). These data suggest that α_1D-adrenergic agonists can release ATP from lacrimal gland pieces. 

Figure 2.

 

Effect of phenylephrine on ATP release from lacrimal gland pieces. Time-dependent release of ATP from lacrimal gland pieces stimulated with α_1D-adrenergic agonist phenylephrine (10⁻⁴ M) for 0 to 10 minutes (A). Pieces were preincubated with the α_1D-adrenergic receptor antagonist BMY 7378 (10⁻⁴ M) for 30 minutes before stimulation with Ph (10⁻⁴ M) for 30 seconds (B). Values are mean ± SEM of three independent experiments. *Significant difference from no additions. #Significant difference between agonist alone and agonist plus inhibitor.

A second method was used to confirm that α_1D-adrenergic agonists stimulated ATP release. Isolated acini were preincubated with apyrase, which breaks down extracellular ATP into adenosine monophosphate, which can be converted to adenosine and should thus prevent α_1D-AR agonist activation of P2X₇ receptors (Table 1). Apyrase completely blocked the phenylephrine-stimulated increase in [Ca²⁺]_i from acinar cells (Figs. 3A, 3B). We conclude that α_1D-adrenergic agonists release ATP from the lacrimal gland. The released ATP then would activate P2X₇ receptors to increase [Ca²⁺]_i. 

Figure 3.

 

Effect an inhibitor of ATPases, apyrase, on [Ca²⁺]_i stimulated by phenylephrine in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing fura-2 with α_1D-adrenergic agonist phenylephrine (10⁻⁵ M) after 30-minute preincubation with apyrase (20 U/mL). Traces are mean of five experiments (A). Mean change in peak [Ca²⁺]_i from five experiments (B). Values are mean ± SEM. Arrow: time of addition of phenylephrine. *Significant difference from no additions (0). #Significant difference between agonist alone and agonist plus inhibitor.

α_1D-Adrenergic and P2X₇ Receptor Antagonists Cross-Inhibit Each Other's Intracellular Ca²⁺ Response in Lacrimal Gland Acinar Cells

Activation of α_1D-AR and P2X₇ receptors increases [Ca²⁺]_i in lacrimal gland acini. To determine whether these signaling pathways interact, we stimulated fura-2–loaded acini with the P2X₇ selective agonist BzATP after incubation with and without the α_1D-adrenergic receptor inhibitor BMY 7378 at 10⁻⁴ M (Table 1). BzATP at 10⁻⁴ M increased the [Ca²⁺]_i by 123.0 ± 32.5 nM (Figs. 4A, 4B). BMY 7378 significantly inhibited the response to 35.4 ± 12.0 nM. As a positive control, the α_1D-AR agonist phenylephrine increased [Ca²⁺]_i by 157.8 ± 26.0 nM. BMY 7378 at 10⁻⁴ M significantly reduced the phenylephrine response by 79%. These results suggest that the inhibition of α_1D-AR blocks P2X₇ receptor stimulation. 

Figure 4.

 

Effect of α_1D-adrenergic agonist antagonist BMY 7378 and P2X₇ receptor antagonist A438079 on [Ca²⁺]_i in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing fura-2 with BzATP (10⁻⁴ M) or α_1D-adrenergic agonist phenylephrine (10⁻⁵ M) after 30-minute preincubation with BMY 7378 (10⁻⁴ M). Traces are mean of five (phenylephrine) or six (BzATP) experiments (A). Mean change in peak [Ca²⁺]_i from five (phenylephrine) or six (BzATP) experiments (B). Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing fura-2 with α_1D-adrenergic agonist phenylephrine (10⁻⁵ M) after 30-minute preincubation with A438079 (10⁻⁴ M) (C). Trace is the mean of seven experiments. Mean change in peak [Ca²⁺]_i from seven experiments (D). Values are mean ± SEM. Arrows: time of addition of agonists. *Significant difference from no addition. #Significant difference between agonist alone and agonist plus antagonist.

In separate experiments, cells were stimulated with the α_1D-adrenergic agonist phenylephrine (10⁻⁵ M) with and without preincubation with the P2X₇ antagonist A438079 (10⁻⁴ M; Table 1). Phenylephrine increased [Ca²⁺]_i by 146.8 ± 28.5 nM (Figs. 4C, 4D). A438079 significantly decreased the response to 73.8 ±17.0 nM. These data imply that the inhibition of P2X₇ receptors blocks the stimulation of α_1D-AR. 

Taken together these data suggest that P2X₇ and α_1D-AR can activate each other to increase [Ca²⁺]_i. 

Activation of α_1D-AR and P2X₇ Receptors Use Separate Cellular Signaling Pathways to Increase [Ca²⁺]_i in Lacrimal Gland Acini

[Ca²⁺]_i was measured in fura-2–loaded acini stimulated simultaneously with phenylephrine and BzATP at maximal and submaximal concentrations for increasing [Ca²⁺]_i. Phenylephrine (10⁻⁵ M) and BzATP (10⁻⁴ M), at maximal concentrations, each significantly increased [Ca²⁺]_i to 161.7 ± 40.0 nM and 258.2 ± 29.1 nM, respectively (Figs. 5A, 5B). When the two were added at the same time, the increase in [Ca²⁺]_i was 315.4 ± 67.6 nM, which was lower than, but not significantly different from, the calculated additivity. Similar results were obtained when a submaximal concentration of phenylephrine was used, but the difference in the calculated and experimentally obtained increase in [Ca²⁺]_i was statistically significant (Figs. 5C, 5D). 

Figure 5.

 

Effect of simultaneous addition of phenylephrine and BzATP on [Ca²⁺]_i in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by the stimulation of lacrimal gland acini containing fura-2 with α_1D-adrenergic agonist phenylephrine (10⁻⁵ M or 10⁻⁶ M) or BzATP (10⁻⁴ M) or the two added simultaneously. Traces are the mean of seven experiments (A, C). Mean change in peak [Ca²⁺]_i from seven experiments (B, D). Values are mean ± SEM. (arrows) Addition of agonists. *Significant difference from no additions (0). #Significant difference of experimental additivity from calculated additivity. n.s., no significant difference.

A second experimental paradigm was used to further explore the signaling pathways used by α_1D-AR and P2X₇ receptors to increase [Ca²⁺]_i. Instead of adding the two agonists simultaneously, the addition of one agonist preceded the second. Maximal concentrations of phenylephrine (10⁻⁵ M) and BzATP (10⁻⁴ M) were used. When phenylephrine was used first, it increased the [Ca²⁺]_i by 170.4 ± 55.4 nM (Figs. 6A, 6B). When phenylephrine was used after BzATP, the response was not altered, and the increase in [Ca²⁺]_i was 183.9 ± 82.7 nM. Use of BzATP first increased the [Ca²⁺]_i to 232.5 ± 31.7 nM. When BzATP was used after phenylephrine, the response was not altered and the increase in [Ca²⁺]_i was 276.0 ± 116.2 nM. 

Figure 6.

 

Effect of sequential addition of phenylephrine and BzATP on [Ca²⁺]_i in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing fura-2 with α_1D-adrenergic agonist phenylephrine (10⁻⁵ M; first arrow) followed by BzATP (10⁻⁴ M; second arrow) or the reverse. Traces are mean from four experiments (A). Mean change in peak [Ca²⁺]_i from four experiments (B). Values are mean ± SEM.

These results taken together suggest that when maximal concentrations of agonists were used P2X₇ and α_1D-AR receptors used different cellular mechanisms to increase [Ca²⁺]_i. When the α_1D-adrenergic agonist was decreased, the pathways were no longer additive. This could have reflected the use of an overlapping pathway when the agonist concentration was altered. 

α_1D-Adrenergic, but Not P2X₇, Receptors Use PKC to Increase [Ca²⁺]_i

Acini containing fura-2 were stimulated with phenylephrine (10⁻⁵ M) or BzATP (10⁻⁴ M) that had been preincubated with and without the PKC inhibitor calphostin C (Table 1). Phenylephrine increased [Ca²⁺]_i to 241 ± 61.2 nM (Figs. 7A, 7C). Preincubation with calphostin C at 10⁻⁸ and 10⁻⁷ M significantly decreased [Ca²⁺]_i to 88.3 ± 22.1 nM and 65.8 ± 24.1 nM, respectively. BzATP (10⁻⁴ M) increased the intracellular [Ca²⁺] by 119.0 ± 30.8 nM (Figs. 7B, 7C). Preincubation with calphostin C at either concentration did not alter the BzATP-induced Ca²⁺ response. Thus, stimulation of α_1D-adrenergic, but not of P2X₇, receptors activates PKC isoforms to alter the [Ca²⁺]_i. Differential activation of PKC isoforms by α_1D -adrenergic and P2X₇ agonists is consistent with the use of separate signaling pathways by these two agonists to increase [Ca²⁺]_i. 

Figure 7.

 

Effect of inhibition of PKC on phenylephrine- and BzATP-stimulated [Ca²⁺]_i in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing fura-2 with α_1D-adrenergic agonist phenylephrine (10⁻⁵ M) or BzATP (10⁻⁴ M) after 30-minute preincubation with calphostin C (CalC). Traces are the mean of seven experiments for phenylephrine (A), and traces are the mean of four experiments for BzATP (B). Mean change in peak [Ca²⁺]_i from seven experiments (phenylephrine) and four experiments (BzATP) (C). Values are mean ± SEM. Arrows: addition of agonists. *Significant difference from no additions (0). #Significant difference between agonist alone and agonist plus inhibitor.

Activation of Ca²⁺/Calmodulin-Dependent Protein Kinase Does Not Alter the P2X₇ Receptor-Induced Increase in [Ca²⁺]_i in Lacrimal Gland Acini

Because P2X₇ receptors have been reported to activate Ca²⁺/calmodulin-dependent protein kinase (CaMK) to increase pore formation, ¹⁴ we next investigated whether stimulation of the P2X₇ receptor activates CaMK to increase [Ca²⁺]_i in rat lacrimal gland. Acini were preincubated with or without the CaMKII inhibitor CSG 9343B (10⁻⁵ and 10⁻⁴ M) and then were stimulated with phenylephrine (Table 1). Phenylephrine significantly increased [Ca²⁺]_i by 198.0 ± 53.2 nM. Preincubation with CSG 9343B (10⁻⁴ M) significantly decreased the phenylephrine-induced response to 61.8 ± 19.9 nM (Figs. 8A, 8B). BzATP significantly increased [Ca²⁺]_i by 233.6 ± 40.7 nM (Figs. 8C, 8D). Inhibition of CaMKII with CSG, used at 10⁻⁶ to 10⁻⁴ M, did not alter the BzATP-induced increase in [Ca²⁺]_i at any concentration. Thus, P2X₇ receptors do not appear to activate CaMKII to increase [Ca²⁺]_i. 

Figure 8.

 

Effect of inhibition of calcium/calmodulin kinase II (CamKII) on phenylephrine- and BzATP-stimulated [Ca²⁺] in lacrimal gland acini. Time-dependent increase in [Ca²⁺]_i induced by stimulation of lacrimal gland acini containing fura-2 with α_1D-adrenergic agonist phenylephrine (10⁻⁵ M; A) or BzATP (10⁻⁴ M, C) after 30-minute preincubation with the CaMKII inhibitor CSG 9343B. Traces are the mean of four experiments. Mean change in peak [Ca²⁺]_i from Ph (B) and BzATP (D) are from four experiments. Values are mean ± SEM. Arrows: addition of agonists. *Significant difference from agonist alone.

Differential activation of CaMKII by α_1D -adrenergic and P2X₇ agonists is consistent with the use of separate signaling pathways by these two agonists to increase [Ca²⁺]_i. 

α_1D-Adrenergic and P2X₇ Receptor Inhibitors Do Not Cross-Inhibit Each Other's Secretory Response in Lacrimal Gland Acinar Cells

Because α_1D-adrenergic agonists release ATP to activate P2X₇ receptors and increase the [Ca²⁺]_i, we determined whether this effect was extended to the entire signaling pathway, resulting in protein secretion. Lacrimal gland acini were stimulated with BzATP at 10⁻⁴ M, and protein secretion was measured. BzATP significantly stimulated protein secretion by 1.5 ± 0.1-fold (Fig. 9A). Preincubation with BMY 7378 (10⁻⁴ M) did not affect BzATP-stimulated secretion. In analogous experiments, acini were stimulated with the α_1D-adrenergic agonist phenylephrine at 10⁻⁴ M, which increased secretion by 2.7 ± 0.5-fold (Fig. 9B). Preincubation with the P2X₇ inhibitor A438079 (10⁻⁴ M) did not alter phenylephrine-stimulated secretion. In contrast to the increase in [Ca²⁺]_i, α_1D-AR or P2X₇ receptor inhibitors did not block protein secretion stimulated by the other agonist. 

Figure 9.

 

Effect of α_1D-adrenergic and P2X₇ receptor antagonists on protein secretion stimulated by phenylephrine or BzATP from lacrimal gland acini. Peroxidase secretion, our index of protein secretion, was measured from lacrimal gland acini preincubated with the α_1D-adrenergic receptor antagonist BMY 7378 (10⁻⁴ M) for 30 minutes and then stimulated with BzATP (10⁻⁴ M) for 40 minutes (A) or preincubated with the P2X₇ receptor antagonist A438079 (10⁻⁴ M) for 30 minutes and then stimulated with phenylephrine (phenylephrine) (10⁻⁴ M) for 40 minutes (B). Values are the mean ± SEM of four independent experiments. *Significant difference from no additions.

α_1D-Adrenergic and P2X₇ Receptor Agonists Use Separate Cellular Pathways to Stimulate Protein Secretion from Lacrimal Gland Acini

Protein secretion was measured from acini stimulated simultaneously with phenylephrine (10⁻⁴ M) and BzATP (10⁻⁴ M) at concentrations maximal for secretion. Phenylephrine and BzATP each significantly increased protein secretion by 1.7 ± 0.3- and 1.2 ± 0.1-fold respectively (Fig. 10A). When the two agonists were added simultaneously, the increase in protein secretion was not significantly different from the calculated additive response. Similar results were obtained when this experiment was performed in the absence of Mg²⁺ to increase the P2X₇ receptor response (Fig. 10B). These results suggest that, similar to the increase in [Ca²⁺]_i, α_1D-AR and P2X₇ receptors use different cellular pathways to stimulate protein secretion. 

Figure 10.

 

Effect of simultaneous addition of phenylephrine and BzATP on protein secretion from lacrimal gland acini. Peroxidase secretion, our index of protein secretion, was measured from lacrimal gland acini stimulated for 40 minutes with phenylephrine (10⁻⁴ M), BzATP (10⁻⁴ M), or the two agonists added simultaneously. The calculated amount of secretion from the two agonists added simultaneously is also plotted. Results obtained in the presence (A) and the absence (B) of extracellular Mg²⁺. Values are the mean ± SEM of four independent experiments. *Significant difference from no addition. n.s., no significant difference.

Discussion

In the present study we found that activation of α_1D-AR releases ATP from acini and stimulates P2X₇ receptor activity to increase [Ca²⁺]_i, but not to stimulate protein secretion. Similarly, P2X₇ receptors stimulate α_1D-AR activity to increase [Ca²⁺]_i but not to stimulate protein secretion. These results are supported by three different types of experiments. First, α_1D-AR releases ATP from pieces. Second, the removal of extracellular ATP with apyrase blocks α_1D-AR stimulation of P2X₇ receptors. Third, blocking α_1D-AR receptors with the α_1D-AR antagonist prevents the P2X₇ receptor from increasing [Ca²⁺]_i and, conversely, inhibiting P2X₇ receptors with a P2X₇ receptor antagonist blocks α_1D-AR from increasing [Ca²⁺]_i. Interestingly, this cross-stimulation does not extend to protein secretion. The interaction of the two receptors to increase [Ca²⁺]_i could be a direct interaction, suggesting that the two receptors are part of a signaling complex such as a signalosome. In support of the possible direct interaction of receptors, in myenteric neurons, nicotinic acetylcholine and P2X₂, P2X₃, and P2X₄ receptors are close enough so that a conformational change in one induced by activation can inhibit the other receptor. ^15,16 In the lacrimal gland, it is possible that α_1D-AR and P2X₇ receptors are colocalized in a complex so that inhibition of P2X₇ and α_1D-AR could affect the activation of the other. It is also possible that the two receptors regulate one another's signaling components within the signalosome. ¹⁷ In addition, Michel et al. ¹⁸ showed that different inhibitors of the P2X₇ receptor have negative and positive allosteric effects on these receptors and, thus, could change the P2X₇ receptor conformation so that it could alter the α_1D-AR. 

Both α_1D-AR and P2X₇ receptors have extracellular and intracellular domains that can be modified to change the way the receptor increases [Ca²⁺]_i. ¹⁹ For example, the intracellular tail of the P2X₇ receptor contains consensus sites for PKC phosphorylation. In addition the rat, but not the human, P2X₇ receptor has a calmodulin-binding domain in its C-terminal ²⁰ that prevents its desensitization with repeated agonist applications. P2X₇ receptors also have a Src homology 3 death domain in the C terminus, ²¹ which is involved in P2X₇ receptor activation of pannexin1, resulting in pore formation. Findings from a previous study ¹³ along with those in the present study indicate that neither stimulation nor inhibition of PKC activity appears to alter the P2X₇ receptor increase in [Ca²⁺]_i. ¹⁹ In the design of the present study, the activation of CaMKII did not alter the single addition of BzATP to increase in [Ca²⁺]_i. Finally, we did not test the role of Src in the present study. Thus, to date, the mechanism used by α_1D-AR to stimulate P2X₇ receptors to increase in [Ca²⁺]_i and vice versa has not been identified. 

Given that P2X₇ and α_1D-AR stimulate each other, it might be expected that these two receptors activate a common signaling pathway to increase [Ca²⁺]_i and to stimulate secretion. Thus, with the sequential addition of P2X₇ and α_1D-AR agonists, the agonist added first would decrease the response of the second. This was not the case in the lacrimal gland. Both simultaneous and sequential addition of P2X₇ and α_1D-AR agonists caused additive [Ca²⁺]_i responses, and simultaneous addition of both agonists caused additive secretory responses. Furthermore, inhibitors of P2X₇ receptors and α_1D-AR did not block the secretion stimulated by the other agonist. The results indicate that P2X₇ and α_1D-AR use different cellular mechanisms to increase [Ca²⁺]_i and to stimulate secretion. P2X₇ receptors are nonselective ion channels that increase [Ca²⁺]_i. The mechanism that the activation of α_1D-AR uses to increase [Ca²⁺]_i in rat lacrimal gland acini is unknown. It is known that α_1D-AR does not induce the production of InsP₃ in rats, though α_1D-AR uses this compound to increase [Ca²⁺]_i in mouse lacrimal gland acini. ^3,22 Inhibition of PKC and CaMK in turn inhibited α_1D-AR, but not P2X_7, receptor-stimulated increases in [Ca²⁺]_i, indicating that P2X₇ and α_1D-AR use separate mechanisms to increase intracellular calcium, thus accounting for the additivity of the increase in [Ca²⁺]_i when both receptors are activated. 

In the present study, P2X₇ and α_1D-AR antagonists cross-inhibited the increase in [Ca²⁺]_i caused by the other receptor but did not block protein secretion. These differential effects on [Ca²⁺]_i compared with secretion suggest that increasing [Ca²⁺]_i is not the major mechanism by which α_1D-AR stimulates protein secretion. The results also imply that the α_1D-AR and P2X₇ receptor-mediated increase in [Ca²⁺]_i is regulated by different components of the signalosome than are the components that cause secretion. 

Results of the present study showed that protein secretion, similar to [Ca²⁺]_i, increased by the simultaneous addition of P2X₇ and α_1D-AR agonists was additive, suggesting that in the lacrimal gland α_1D-AR and P2X₇ receptors activate different signaling pathways to stimulate protein secretion. In lacrimal gland acini, α_1D-AR activate eNOS/cGMP, increase PKCε activity, and raise the [Ca²⁺]_i by an unidentified mechanism to stimulate secretion. ^{4 –6} To attenuate secretion α_1D-AR agonists transactivate the EGF receptor to induce the ERK1/2 cascade and activate PKCα and PKCδ. ^5,7 Signaling pathways used by P2X₇ receptors in lacrimal acini have been less well studied. A previous publication showed that P2X₇ receptors increase the [Ca²⁺]_i using the ionotropic receptor itself to stimulate secretion and activate ERK1/2, which could attenuate secretion. ¹² In a previous study, ¹³ as in the present study, an effect of P2X₇ receptors on PKC isoforms and CaMKII was ruled out. To date, the signaling pathways used by α_1D-AR and P2X₇ receptors differ in their use of PKC isoforms. Further research is warranted on the signaling mechanisms used by P2X₇ receptors to stimulate lacrimal gland secretion to identify the different signaling mechanisms used by α_1D-AR and P2X₇ receptors. 

In the lacrimal gland, M₃AchR and α_1D-AR use separate different mechanisms to stimulate protein secretion. ^4,5,8 The interaction of the M₃AchR and α_1D-AR with P2X₇ receptors supports this concept. Compared with the signaling pathways used by α_1D-AR, described in the previous paragraph, to stimulate the secretion, M₃AchR activates PLCβ, producing InsP₃ that increases the [Ca²⁺]_i and producing diacylglycerol that activates PKCα, PKCδ, and PKCε. ^3,5 To attenuate secretion, M₃AchR induces PLD that, using Rho and ROCK, stimulates ERK1/2, and it induces the nonreceptor tyrosine kinases Pyk2 and Src to transactivate the EGFR, thereby activating the ERK1/2 cascade. ⁹ Even though there is some overlap in the signaling components, distinct signaling pathways are activated by the two types of receptors. In the same type of paradigm, M₃AchR and α_1D-AR interact differently with P2X₇ receptors. M₃AchR activates P2X₇ receptors to increase the [Ca²⁺]_i and to stimulate secretion. M₃AchR releases ATP from nonacinar cells that could activate P2X₇ receptors and probably use a direct intracellular pathway to stimulate the activity of P2X₇ receptors. ¹³ In contrast, α_1D-AR release ATP from acini to activate P2X₇ receptors and, in turn, the activation of P2X₇ receptors stimulates α_1D-AR to increase [Ca²⁺]_i. The signaling mechanisms used by α_1D-AR and P2X₇ receptors to increase the [Ca²⁺]_i appear to be different, but those used by the M₃AchR and P2X₇ receptors overlap. However, the signaling mechanisms used by α_1D-AR, M₃AchR, and P2X₇ receptors to stimulate protein secretion are different. Thus, to date, the cellular mechanisms used by M₃AchR and α_1D-AR to induce P2X₇ receptors activity appear to be different. 

In one other tissue, the interaction of αAR and P2X₇ receptors has been investigated. In hypothalamo-neurohypophyseal explants, the α_1D-AR agonist causes oxytocin and vasopressin secretion, and co-exposure of α₁-AR agonists with ATP prolongs and potentiates the secretory response. ^23,24 Gomes et al. ²⁴ found that ATP activates P2X₂ receptors that recruit P2X₃ and P2X₇ receptors, which are responsible and desensitization resistant and thereby prolong secretion. ²⁴ In contrast, in the present study, P2X₇ receptors and α_1D-AR do not appear to potentiate the secretory response. 

We conclude that in the lacrimal gland, the activation of α_1D-AR releases ATP that induces P2X₇ receptors to increase [Ca²⁺]_i, but not to stimulate protein secretion. P2X₇ receptors, in turn, activate α_1D-AR to increase [Ca²⁺]_i, but not to stimulate protein secretion. Furthermore, α_1D-AR compared with P2X₇ receptors use different cellular mechanisms to increase [Ca²⁺]_i and to cause protein secretion. The interaction of P2X₇ receptors with α_1D-AR is different from the interaction of P2X₇ receptors with M₃AchR. 

Footnotes

 Supported by National Institutes of Health Grant EY06177.

Footnotes

 Disclosure: D.A. Dartt, None; R.R. Hodges, None