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Am J Physiol Regul Integr Comp Physiol 292: R423-R431, 2007. First published September 14, 2006; doi:10.1152/ajpregu.00495.2006
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

Zhilin Song, Sukumar Vijayaraghavan, and Celia D. Sladek

Department of Physiology and Biophysics, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado

Submitted 13 July 2006 ; accepted in final form 13 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ATP increases intracellular calcium concentration ([Ca2+]i) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca2+-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca2+]i in dissociated SON neurons. However, the ATP-induced increase in [Ca2+]i persisted after removal of Ca2+ from the perifusate ([Ca2+]o). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca2+ release from intracellular stores, whereas P2XRs are Ca2+-permeable ion channels. Depletion of [Ca2+]i stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca2+]i in zero, but not in 2 mM [Ca2+]o, indicating that both Ca2+ influx and release of intracellular Ca2+ contribute to the ATP response. Ca2+ influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca2+ channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca2+]i. Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca2+]i (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca2+]i than UTP or UDP. MRS2179, a specific P2Y1R antagonist, eliminated the response to ATP in zero [Ca2+]o. Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca2+]i, and the P2Y1R subtype is more prominent than P2Y2R, P2Y4R, or P2Y6R in SON.

calcium imaging; fura-2 acetoxymethyl ester; thapsigargin; pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid; cadmium

ATP ACTS AS A NEUROTRANSMITTER in the central and peripheral nervous system. It is copackaged with other neurotransmitters in synaptic vesicles, coreleased during exocytosis of those vesicles, and activates purinergic receptors (PR) located both pre- and postsynaptically (3, 9). There are two major classes of PRs. P2X purinergic receptors (P2XRs) are ligand gated, nonselective cation channels that are highly permeable to Ca2+. P2Y purinergic receptors (P2YRs) are G protein-coupled receptors. Multiple subtypes of both classes of receptors have been cloned and classified as P2X1–7 and P2Y1–8 (19). Both classes of receptors can increase intracellular calcium concentration ([Ca2+]i). Since P2XRs are Ca2+-permeable, ligand-gated ion channels, their activation initiates Ca2+ influx through the channel. The channel is also permeable to Na+, which causes membrane depolarization, allowing Ca2+ influx through voltage-sensitive Ca2+ channels. P2YRs are G{alpha}q/11-coupled receptors and thus activate inositol triphosphate (IP3), which, in turn, releases Ca2+ from intracellular stores.

ATP has been shown to increase vasopressin (VP) and oxytocin (OT) release from hypothalamo-neurohypophyseal system (HNS) explants (13) and to excite supraoptic nucleus (SON) neurons in vivo and in vitro (6, 11, 21). It is thought to serve as a prominent cotransmitter in the A1 catecholamine pathway that transmits information about hypovolemia and hypotension to the SON and paraventricular nucleus, because, although selective destruction of the A1 pathway prevents activation of SON neurons by hypovolemia, antagonism of PRs but not adrenergic or excitatory amino acid receptors inhibited A1-induced excitation of SON neurons (5, 6). The potential importance of ATP as a cotransmitter in this pathway is suggested by the dramatic potentiation of VP and OT release observed when ATP is applied to HNS explants in conjunction with an adrenergic agonist (13). Combined application of these agents converts the small transient increases in VP and OT release observed in response to ATP and phenylephrine alone to a large increase in hormone release that is sustained for hours.

P2XRs have been implicated in these responses, because, at 10 µM, a concentration that selectively antagonizes P2XRs, PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid) blocked ATP-induced VP and OT release from HNS explants (13); the P2XR agonists, {alpha},beta-methylene-ATP, beta,{gamma}-methylene-ATP, and 2-methylthio-ATP, mimicked the depolarizing effects of ATP on SON neurons (11); mRNA for several P2XR subtypes was identified in SON by RT-PCR and in situ hybridization (21); and ATP-induced increases in [Ca2+]i were abolished by removing extracellular Ca2+ concentration ([Ca2+]o) from dissociated SON neurons during imaging of the Ca2+-sensitive dye, fura-2 AM (21). However, in the current experiments, we obtained evidence for ATP activation of P2YRs as well as P2XRs in SON neurons. ATP induced Ca2+ release from intracellular stores, as well as influx of [Ca2+]o, and P2YR agonists increased [Ca2+]i. This is important because it may underlie integration of osmotic and hemodynamic regulation of VP release, and it is important for understanding the mechanisms underlying synergistic responses to ATP and other neurotransmitters by neurons in SON and other areas of the central nervous system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male Sprague-Dawley rats [Crl:CD(SD)Br; Charles Rivers Laboratories, Wilmington, MA] were used in all of the experiments. All protocols used were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Colorado at Denver and Health Sciences Center.

Calcium Imaging

Dye loading. The HNS explant preparation was prepared from 125- to 150-g male Sprague-Dawley rats. It includes the VP and OT neurons of the SON with their axons and terminals in posterior pituitary. This explant was used for live cell Ca2+ imaging, as described previously (23). The AM ester form of fura-2 (Molecular Probes, Carlsbad, CA; 50 µg) was dissolved in 50 µl of anhydrous dimethyl sulfoxide to make a 1 mM stock solution. Explants were incubated in 100 µM fura-2 AM for 10 min followed by a 50-min incubation in 20 µM fura-2 AM in medium containing 0.02% pluronic acid F-127 (Molecular Probes). Dye loading and imaging were performed in F12 nutrient medium (Sigma), fortified with 1 mg/ml glucose and 1.7 mM CaCl2, and gassed with 95% O2–5% CO2.

Perifusion and drug delivery. Explants were placed in a chamber fitted onto the stage of an upright fluorescent microscope. The chamber was formed by an adaptor (Bioscience Tools, San Diego, CA) placed in a 35-mm Corning petri dish, whose base was coated with Sylgard (1 mm). The explant was stabilized with a weighted, ring-shaped stainless steel net, which was pinned onto the Sylgard base. Gassed medium was delivered by a gravity-driven eight-valve system (Warner Instruments, Hamden, CT) to the perifusion port at a rate of ~3 ml/min and was removed by a vacuum port at the opposite side.

Fluorescence ratio imaging. The explant was positioned in the recording chamber with its ventral side up, stabilized with a weighted nylon mesh, and allowed to equilibrate for 1 h. Thus the optic chiasm is easily visualized, and SON can be located anatomically in the tissue immediately rostral and parallel to optic chiasm. Fura-2-loaded magnocellular neurons were alternatively excited with 340-nm and 380-nm UV light from a Xenon source (Sutter Instruments, Novato, CA). Exposure time for 380-nm wavelength was between 200 and 500 ms and was tripled for 340-nm wavelength accordingly. Emitted light was passed through a x60 fluor water immersion lens attached to an Olympus upright microscope and collected at 510 nm by an intensified charge-coupled device camera (Hamamatsu, Japan). Paired 340- and 380-nm excitation images were acquired every 3 s using SlideBook software (Intelligent Imaging, Denver, CO) for a period of 100 frames. Explants treated with 10 µM ionomycin to allow [Ca2+]i to equilibrate with [Ca2+]o gave an apparent maximum ratio (Rmax) of 1.0684 ± 0.001 (n = 15) in 0.3 mM Ca2+ and 2.0643 ± 0.0028 (n = 16) in 2 mM Ca2+. As both values far exceed the highest ratio achieved by all agents studied in all experimental preparations, the peak responses reported were not limited by the maximum detectable change in the 340 nm-to-380 nm (340/380) ratio. The ratio data are presented as percentage of the basal 340/380 ratio for each cell (see below).

Data analysis. Magnocellular neurons were identified by the size of the cell body (>25 µm in diameter), their location, and for some as described previously (23), their response to VP. Data from the identified neurons were collected as the average ratio of fluorescence from 340-nm excitation over fluorescence from 380-nm excitation from a region of interest that included the complete area of the cell body, including the nucleus, if it was (rarely) evident at the focal plane of the image. This ratio is a direct, linear measurement of [Ca2+]i within physiological ranges (10). A baseline ratio value for each neuron was determined by averaging ratios of the 10 frames preceding drug application. Responses were then expressed as percentage changes in 340/380 ratio relative to basal. The means ± SE of the percentage values from individual neurons were calculated and plotted. Parametric one-way ANOVA (F ratio) or Kruskal-Wallis one-way ANOVA on ranks (H value) were used to determine whether there were significant group differences in the peak responses. All experiments were replicated in two preparations. ANOVA was used to compare the responses between the two explant preparations in representative experiments and demonstrated that the responses were not statistically different. Therefore, additional replications were not performed.

Drug interactions with fura-2. The P2XR antagonists (PPADS, iso-PPADS, and suramin) and cadmium, a blocker of voltage-gated Ca2+ channels, altered the baseline 340/380 ratio of fura 2 fluorescence in SON neurons. To evaluate the cause of this and to determine whether fura-2 could be used to assess changes in intracellular Ca2+ in the presence of these agents, the effect of the agents on fura-2 340-nm and 380-nm induced emissions were analyzed in a cuvette-based assay using a QM-6/2003 fluorometer (Photon Technology International, Birmingham, NJ) using the fura-2 pentapotassium salt (45 µM). The assay was validated by the demonstration that the fluorometer detected the anticipated changes in 340 nm, 380 nm, and the 340/380 ratio when the Ca2+ concentration was raised to 10 mM or when EGTA was added to the buffer to render it Ca2+ free (Table 1). Also, 2 mM Mn2+, which is known to quench fura-2 fluorescence at both wavelengths, reduced both the 340-nm and 380-nm evoked emissions to barely detectable. Note, however, that, despite intense quenching, the 340/380 ratio in 2 mM Mn2+ was slightly greater than control (114%) due to the 340-nm signal being slightly greater than the 380-nm signal. PPADS also quenched both 340-nm and 380-nm evoked emissions. At 100 µM, the effect of PPADS on 380 nm was much greater than that on 340 nm, resulting in a substantial increase in the 340/380 ratio, but, at 10 µM, the difference is much smaller so that the 340/380 ratio is only slightly increased. This concentration of PPADS and iso-PPADS was used in our current experiments. The effect of suramin was limited to the 340-nm evoked emission and, therefore, resulted in a quite large decrease in the 340/380 ratio at the concentration deemed effective to block P2 purinergic receptors. As a result, the remaining Ca2+-induced decrease in 380-nm emission was small and not a reliable indication of changes in [Ca2+]i. Therefore, the effect of suramin was not assessed in our current experiment. Cadmium (Cd2+, 200 µM) behaved like Ca2+, increasing the 340-nm and decreasing the 380-nm evoked emissions and resulting in a large increase in the ratio. The flourometric assay clearly demonstrated that direct interaction of the antagonists with fura-2 results in differential and selective quenching of either 340-nm or 380-nm emission. This suggests that, in the current experiments, the cells were permeable to the drugs, thereby allowing the antagonists to directly interact with the dye inside the cells. This has not been reported in other studies (21, 25), but it may reflect differences in the dye-loading protocols used in isolated or cultured cell preparations compared with the organotypic preparation used in this study. Despite the impact of these agents on fura emission, we were able to demonstrate qualitative changes in the response to ATP in the presence of PPADS and cadmium (see RESULTS).


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  		Table 1. Interaction of agents with fura-2

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Source of ATP-induced [Ca2+]i: Effect of Calcium-Free Medium, Thapsigargin Pretreatment, and/or Cadmium

ATP induces a prompt and prominent increase in [Ca2+]i in SON neurons studied in organotypic HNS explants (Fig. 1A). To identify the source of this increase in [Ca2+]i, the response to ATP was examined in a calcium-free medium (Fig. 1B). Although the response to ATP was attenuated, ATP still induced an increase in [Ca2+]i in the absence of [Ca2+]o. Since this result suggests mobilization of Ca2+ from intracellular stores, the effect of ATP was examined following pretreatment of HNS explants with thapsigargin (200 nM, 0.5 h) to deplete intracellular Ca2+ stores. As shown in Fig. 1C, thapsigargin pretreatment also attenuated, but did not abolish, the ATP-induced increase in [Ca2+]i. The response was absent in thapsigargin-pretreated explants studied in Ca2+-free medium (Fig. 1D). As expected, switching to zero [Ca2+]o following thapsigargin treatment (Fig. 1D) resulted in a decrease in [Ca2+]i, demonstrating the contribution of Ca2+ influx to maintenance of [Ca2+]i when the intracellular stores are depleted.


Figure 1
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  		Fig. 1. Evaluation of the source of the ATP-induced increase in intracellular calcium concentration ([Ca2+]i). A: time course of the response to the addition of ATP (200 µM) (arrow) in 2 mM Ca2+ (ATP.NCa). B: ATP response following removal of extracellular Ca2+ (ATP.0Ca). Ca2+ was removed from the perifusate 10 min before the recording. C: effect of pretreatment with thapsigargin (TG) for 0.5 h during the 1-h equilibration period. TG was washed out 10 min before the recording (ATP.TG.NCa). D: effect of ATP in zero calcium following TG pretreatment (ATP.TG.0Ca). The effect of removing extracellular Ca2+ on the 340-nm-to-380 nm (340/380) ratio is evident. In all panels, the mean ± SE is plotted for multiple cells (n) recorded in a single representative explant for each treatment. All treatments were replicated in at least 2 explants (see Fig. 2). The basal 340/380 ratio for the 10 frames preceding ATP addition was 0.5145 ± 0.0112 (A), 0.6455 ± 0.0930 (B), 0.7796 ± 0.0068 (C), and 0.5194 ± 0.0116 for (D).

 
These results demonstrate that both Ca2+ influx and release of Ca2+ from intracellular stores contributed to the ATP-induced increase in [Ca2+]i. The peak responses to ATP in Ca2+-replete or Ca2+-free medium, with or without thapsigargin pretreatment, are compared in Fig. 2. The ATP-induced increase in [Ca2+]i was significantly reduced by removal of [Ca2+]o or thapsigargin pretreatment and was obliterated by the combination of thapsigargin pretreatment and Ca2+-free medium (H = 87.306, P < 0.001). These results suggest involvement of both P2XRs and P2YRs, because P2XRs are Ca2+-permeable ion channels, whereas P2YRs are Gq/ll-coupled receptors that induce formation of IP3 that stimulates release of Ca2+ from intracellular stores.


Figure 2
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  		Fig. 2. Comparison of the peak elevation in [Ca2+]i (peak ratio) induced by ATP under the four different intracellular and extracellular Ca2+ conditions illustrated in Fig. 1. a,b,cGroups with different letter designations are statistically different (P < 0.05). Data were obtained from 2 explants per treatment group. In the preparations showing a response, ANOVA demonstrated that the responses were not statistically different between the 2 explant preparations. The total numbers of neurons analyzed per group were as follows: ATP.NCa, n = 30; ATP.0Ca, n = 15; ATP.TG.NCa, n = 25; and ATP.TG.0Ca, n = 32. The mean ± SE basal 340/380 ratio preceding each response was 0.4908 ± 0.0079 for ATP.NCa, 0.6455 ± 0.0930 for ATP.0Ca, 0.7205 ± 0.0153 for ATP.TG.NCa, and 0.4675 ± 0.0119 for ATP.TG.0Ca.

 
Although P2XRs are Ca2+-permeable ionotropic receptors, they mainly flux Na+, resulting in membrane depolarization. This could activate voltage-sensitive Ca2+ channels and thus induce a secondary Ca2+ influx. To evaluate the relative contributions of these two mechanisms to the ATP-induced Ca2+ influx, the response to ATP was analyzed in medium containing 200 µM cadmium to block voltage-sensitive Ca2+ channels, both with and without thapsigargin pretreatment. Although the slow leak of cadmium into the cells resulted in a linear increase over time in the 340/380 ratio recorded in SON neurons (Fig. 3A), the fluorometric assay demonstrated that, at concentrations ≤0.1 µM, cadmium had not saturated the fura, and therefore it did not prevent detection of changes in Ca2+ (Fig. 3B). Thus, as evident in Fig. 3, A and C, if ATP was added during the linear, nonsaturated phase of the cadmium-induced increase in the 340/380 baseline, then changes in the ratio reflective of ATP-induced Ca2+ influx were evident. To evaluate this response, the linear increase in the baseline ratio induced by the leak of cadmium into the cell was subtracted from each response. The resulting peak increase in the 340/380 ratio induced by ATP in the presence of cadmium is shown in Fig. 3D, with and without thapsigargin pretreatment. In the absence of thapsigargin, cadmium reduced the ATP-induced increase in [Ca2+]i by ~50% (Fig. 3, C and D). This response was significantly attenuated when explants were pretreated with thapsigargin to deplete intracellular Ca2+ stores. However, an ATP-induced increase in [Ca2+]i was still detectable (Fig. 3, C and D) in all of the neurons recorded. These results indicate that the ATP-induced influx of [Ca2+]o occurs primarily through voltage-sensitive Ca2+ channels, with a small component coming from another source, presumably the P2XRs. This was further analyzed using PR antagonists.


Figure 3
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  		Fig. 3. Involvement of voltage-gated calcium channels in ATP-induced [Ca2+]i increase. A: several representative traces demonstrating the impact of adding 200 µM cadmium (Cd) to the perifusate on the basal 340/380 ratio and the continued evidence of responses to ATP. B: the effect of cadmium on Ca2+-induced changes in fura-2 fluorescence measured by fluorometer. The 340-nm (red line) and 380-nm (green line) excited emissions and the 340/380 ratio (blue line) as a function of Cd2+ concentration in 0 Ca2+ (open circle) and 1 µM Ca2+ (solid triangle) are shown. Fura-2 potassium (1 µM) was used (10). C: the response to ATP after subtraction of the effect of the cadmium leak on the 340/380 ratio. Time zero indicates when ATP was added to the perifusate. D: comparison of the peak ratio response to 200 µM ATP in the presence of 200 µM cadmium, with (TG.Cd.ATP) or without TG pretreatment (Cd.ATP) (following correction for the cadmium leak). a,b,cGroups with different letter designations are statistically different (H = 45.966, P < 0.001). Data were combined from 2 explants per treatment group, except for the TG.Cd.ATP group, which represents cells in a single explant. The total number of neurons analyzed per group were as follows: ATP, n = 30; Cd.ATP, n = 28; TG.Cd.ATP, n = 16. The mean ± SE basal 340/380 ratio preceding each ATP response was 0.4908 ± 0.0079 for ATP (repeated from Fig. 2), 0.5342 ± 0.0076 for Cd.ATP, and 0.5532 ± 0.0065 for TG.Cd.ATP.

 
PR Subtypes Activated by ATP: Effect of P2XR Antagonists

Effect of PPADS and iso-PPADS with or without thapsigargin pretreatment on ATP-stimulated [Ca2+]i increase were assessed. Ten micromolar PPADS and iso-PPADS were used as specific P2XR antagonists. Although at concentrations ≥30 µM, PPADS can also block some P2YRs, at 10 µM, it is selective for P2XRs. Both PPADS and iso-PPADS increased the basal 340/380 ratio. As shown in Table 1, although PPADS quenched both 340-nm and 380-nm fura-2 emissions, the impact on 340 nm was greater than on 380 nm, resulting in an increase in the ratio. Nevertheless, ATP-induced increases in [Ca2+]i were still evident in the presence of both PPADS and iso-PPADS (Fig. 4, A and C) but were eliminated by thapsigargin pretreatment (Fig. 4, B and D). These results demonstrate that blockade of P2XRs does not eliminate the response to ATP and provide further support for involvement of P2YR in the [Ca2+]i response to ATP. Further experiments were performed using P2YR agonists to evaluate this possibility.


Figure 4
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  		Fig. 4. The effect of pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (or isoPPADS) on the response to ATP with or without TG pretreatment. PPADS (A and B) or isoPPADS (C and D) was administered 3 min before ATP and the 340-to-380 ratio was monitored. TG pretreatment was the same as in Fig. 1. The number of neurons and the average of basal ratios included in these graphs are 25 and 0.6365 ± 0.0102 for A; 16 and 0.6983 ± 0.0132 for B; 16 and 0.5868 ± 0.0126 for C; 18 and 0.7312 ± 0.0132 for D.

 
PR Subtypes Activated by ATP: Effect of P2YR Agonists

Effect of a P2YR agonist cocktail on [Ca2+]i in the presence or absence of TTX. A cocktail of P2YR agonists, consisting of 2-methylthio-ADP (100 µM), UTP (1 mM), and UDP (1 mM), robustly increased [Ca2+]i (Fig. 5, A and C). The presence of TTX (Sigma, 3 µM) did not significantly change the pattern of this response (Fig. 5, A and C). This result supports the hypothesis that P2YRs exist on SON neurons, and their activation increases [Ca2+]i. Further experiments were performed to evaluate the source of the cocktail-induced increase in [Ca2+]i and the relative contribution of individual P2YR subtypes to the response.


Figure 5
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  		Fig. 5. Ca2+ response to the P2Y purinergic receptor (P2YR) agonist cocktail. A: the response in normal Ca2+ perfusion medium in the absence of TTX (P2YR cocktail) or in the presence of TTX (TTX.P2YR cocktail). B: the response in Ca2+-free medium (0Ca.P2YR cocktail) or after TG pretreatment (TG.P2YR cocktail). C: comparison of the peak responses under these conditions. *P < 0.05, normal Ca2+ vs. Ca2+-free medium (ANOVA F = 3.393, P = 0.038). #P < 0.05, normal Ca2+ vs. TG pretreatment (ANOVA H = 76.535, P < 0.001). TTX did not alter the response in normal Ca2+. Data were combined from cells imaged in 2 explants per treatment group, except for the TTX.P2YR cocktail group. The total number of neurons analyzed per group were as follows: P2YR, n = 42; TTX.P2YR, n = 16; 0Ca.P2YR, n = 30; and TG.P2YR, n = 47. The mean ± SE basal 340/380 ratio preceding each response was 0.5448 ± 0.0070 for P2YR, 0.5546 ± 0.0119 for TTX.P2YR, 0.6426 ± 0.0014 for 0Ca.P2YR, and 0.6368 ± 0.0067 for TG.P2YR.

 
Effect of P2YR agonist cocktail on [Ca2+]i in calcium-free medium or after thapsigargin pretreatment. In Ca2+-free medium, the P2YR agonist cocktail induced a large increase in [Ca2+]i (Fig. 5, B and C). The amplitude of the peak response is ~80% of that in normal Ca2+ medium. When intracellular Ca2+ stores were depleted by thapsigargin, the response to the cocktail was markedly attenuated, but a smaller increase in [Ca2+]i persisted, which accounted for ~20% of the total response to the cocktail (Fig. 5, B and C). These results demonstrated that the majority of the [Ca2+]i increase initiated by activation of P2YRs originated from release of intracellular Ca2+ stores, but Ca2+ influx also makes a contribution.

Effect of 2-methylthio-ADP on [Ca2+]i in the presence or absence of TTX. The specific agonist for P2Y1R, 2-methylthio-ADP (100 µM), induced a large increase in [Ca2+]i (Fig. 6). This increase accounted for ~80% of the response to the cocktail. TTX treatment did not change this response significantly (Fig. 6). As is the case for ATP and the P2YR agonist cocktail, essentially every SON neuron responded to 2-methylthio-ADP (Table 2). This result suggests that P2Y1R may be the predominant P2YR subtype in SON.


Figure 6
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  		Fig. 6. Ca2+ response to 2-methylthio-ADP (2-MeS-ADP) in the absence or presence of TTX. A: time course of the responses of cells in a single representative explant for treatments with P2YR agonist cocktail or 2-MeS-ADP in the absence or presence of TTX (TTX.2-MeS-ADP). B: comparison of the peak responses in these three groups (overall ANOVA F = 7.737, P < 0.001). *P < 0.05: P2YR cocktail vs. 2-MeS-ADP, either with or without TTX. Data represent cells combined from 2 explants per treatment group. The total number of neurons analyzed per group were as follows: P2YR cocktail, n = 42; ADP, n = 31; TTX.ADP, n = 48. The mean ± SE basal 340/380 ratio preceding each response was 0.5448 ± 0.0070 for P2YR cocktail, 0.5320 ± 0.0091 for ADP, and 0.5224 ± 0.0101 for TTX.ADP.

 

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  		Table 2. Comparison of the ratio response to P2Y purinergic receptor agonist cocktail, 2-MeS-ADP, UTP, and UDP in the presence or absence of TTX

 
Effect of UTP and UDP on [Ca2+]i in the presence or absence of TTX. In contrast to 2-methylthio-ADP, only 20–36% of recorded neurons responded to UTP or UDP, in the presence of TTX (Table 2), and the peak amplitude to either UTP or UDP was smaller than that to 2-methylthio-ADP or the P2YR agonist cocktail. This suggests that P2Y2R, P2Y4R (UTP is the most potent agonist to these two subtypes), and P2Y6R (for which UDP is the most potent agonist) may also be expressed, although to a lesser extent than P2Y1R, in subpopulations of SON neurons. A significantly larger percentage of neurons (83%) responded to UTP in the absence of TTX, suggesting a role for P2Y2Rs and/or P2Y4Rs in regulating local afferents to SON neurons.

PR Subtypes Activated by ATP: Effect of P2Y1R Antagonist

The possibility that the P2Y1 subtype of P2YRs is primarily responsible for the non-P2XR mediated, ATP-induced increase in [Ca2+]i in SON was further evaluated using a P2Y1R subtype selective antagonist, MRS2179 (2). The ATP-stimulated increase in [Ca2+]i was greatly attenuated by MRS2179 (100 µM, Fig. 7, A and D). The attenuated response in the presence of MRS2179 was not due to the prior exposure to ATP, because robust ATP responses were observed in subsequent experiments in which the order of exposure was reversed (e.g., first ATP + MRS2179 followed by a second exposure to ATP alone). In those experiments, combined treatment with MRS2179 and PPADS further reduced, but did not completely abolish, the [Ca2+]i response to ATP (Fig. 7, B and D), and in Ca2+-free perfusion medium, the response to ATP is barely noticeable in the presence of MRS2179 (Fig. 7, C and D).


Figure 7
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  		Fig. 7. Ca2+ response to ATP in the presence of MRS2179 (MRS), a selective P2Y1R antagonist. A: responses (means ± SE) of the same cells in a representative explant that were treated first with ATP and, after a 30-min wash, challenged with ATP again in the presence of MRS2179. MRS2179 was applied 3 min before ATP. B: this explant was first treated with ATP in the presence of MRS2179 and PPADS (PPADS.MRS). MRS2179 and PPADS were applied 3 min before ATP. After a 30-min wash, the same explant was challenged with ATP again. C: in this experiment, the explant was first treated with ATP in the presence of MRS2179 in Ca2+-free medium (0Ca.MRS). MRS2179 in Ca2+-free medium was applied 3 min before ATP. After 30-min wash with normal Ca2+ medium, the same explant was treated with ATP again. D: comparison of the peak responses under these different conditions (Kruskal-Wallis analysis H = 136.447, P < 0.001). a,b,cGroups with different letter designations are statistically different. Data were combined from 2–3 explants per treatment group. The total number of neurons analyzed per group were as follows: ATP, n = 57; MRS.ATP, n = 44; PP(PPADS).MRS.ATP, n = 33; 0Ca.MRS.ATP, n = 31. The mean ± SE basal 340/380 ratio preceding each response was 0.5306 ± 0.0065 for ATP, 0.5892 ± 0.0058 for MRS.ATP, 0.7094 ± 0.0293 for PP.MRS.ATP, and 0.5060 ± 0.0138 for 0Ca.MRS.ATP.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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Although prior studies on ATP stimulation of VP and OT release by this laboratory (13), as well as Ca2+-imaging experiments in dispersed SON neurons, supported a role for P2XRs in the response to ATP (21), the current studies provide extensive evidence for involvement of P2YRs as well as P2XRs. The expected intracellular signaling cascades initiated by activation of these two receptor types are summarized in Fig. 8. As shown, P2XR activation could induce Ca2+ influx via direct activation of the Ca2+-permeable P2XR and via indirect activation of voltage-sensitive Ca2+ channels. Activation of P2YRs induces release of Ca2+ from intracellular stores as a result of generation of IP3. In the present studies, the conclusion that both P2XRs and P2YRs contribute to the Ca2+ response to ATP is supported by evidence from experiments employing techniques to assess the source of the Ca2+, as well as experiments utilizing selective receptor antagonists and agonists to identify the type of PRs activated. This dual approach is important, because neither approach alone is completely definitive. The receptor antagonists do not block all receptor subtypes and are not completely selective for either P2XR or P2YR subtypes. For example, PPADS blocks the P2X1R, P2X2R, and P2X3R, but not P2X4R, P2X6R, and P2X7R subtypes. Furthermore, in addition to the signal cascades shown in Fig. 8, other factors, such as receptor-operated Ca2+ channels, store-operated Ca2+ channels, and Ca2+-induced Ca2+ release may contribute to the observed changes in [Ca2+]i. Thus the combination of these approaches provides the following strong evidence for involvement of both P2XRs and P2YRs in the ATP-induced Ca2+ response: 1) the ATP-induced increase in [Ca2+]i reflects Ca2+ release from intracellular stores, as well as influx of [Ca2+]o; 2) the P2XR antagonists, PPADS or iso-PPADS alone, do not abolish the ATP-induced increase in [Ca2+]i but do so following thapsigargin pretreatment; 3) P2YR agonists induce an increase in [Ca2+]i; and 4) the Ca2+ response to ATP is eliminated only when both Ca2+ influx and release from stored Ca2+ are blocked by combined exposure to zero external Ca2+ and thapsigargin or when both P2XRs and P2Y1Rs are blocked with PPADS and MRS2179.


Figure 8
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  		Fig. 8. Diagram of possible pathways for ATP to increase [Ca2+]i. 1, ATP activates P2YRs, resulting in inositol triphosphate (IP3) production. IP3 then activates IP3 receptors on the endoplasmic reticulum membrane, resulting in release of stored Ca2+. 2, ATP activates P2XRs, inducing an influx of Ca2+, as well as Na+. 3, The influx of Na+ depolarizes the membrane and activates voltage-gated Ca2+ channels, thus causing a secondary Ca2+ influx. 4, Activation of PKC and/or membrane IP3 receptors induces Ca2+ influx through unidentified receptor-operated Ca2+ channels. {Delta}Vm, depolarization of membrane; DAG, diacylglycerol; PIP2, phosphatidylinositol 4,5-bisphosphate. [Modified from Kapoor and Sladek (13).]

 
The observation that a selective P2Y1R agonist induced a large increase in [Ca2+]i in essentially all SON neurons suggests that these receptors may play a prominent role in regulating the activity of SON neurons and, therefore, the release of VP and OT from the posterior pituitary. This raises the important question of why prior studies did not find evidence for P2YR expression in SON or their involvement in ATP-mediated hormone release. The prior Ca2+-imaging studies were performed in acutely dissociated SON neurons (21). In these studies, the ATP-induced increase in [Ca2+]i was abolished by removal of extracellular fluid Ca2+, indicating that the response was completely dependent on Ca2+ influx. Possible explanations for this difference include the loss of functional P2YRs during the dissociation procedure, potentially as a result of enzymatic damage to the receptors or shearing off of dendritic processes. In the HNS explant preparation used in the present studies, the SON neurons retain their neuritic processes, and the cell membrane is not exposed to enzymatic treatments. In the hormone release studies, the same organotypic explant preparation was used as in the present Ca2+ imaging experiments (23). Therefore, receptor loss by the above mechanisms is unlikely. However, a major difference between the two experiments is the end point studied. In the present experiment, the focus is on the cell body, whereas the end point of hormone release, although it includes activation of perikaryal receptors, is also influenced by regulatory mechanisms at the nerve terminal in the posterior pituitary. ATP acts directly on the nerve terminals and pituicytes in the posterior pituitary. It stimulates VP release from the isolated neural lobe and from isolated neurohypophyseal nerve terminals via a P2X2R (22, 25). Therefore, it is possible that P2XR-mediated mechanisms predominate in the ATP stimulation of hormone release from in vitro preparations in which both hypothalamus and posterior pituitary are exposed to exogenous ATP. Further studies are warranted to evaluate the relative importance of P2XRs and P2YRs in neurohypophyseal hormone release induced by physiological stimuli that release endogenous ATP in SON.

One earlier study did provide evidence for P2YRs in SON (11). This was an electrophysiological study in which 77% of SON neurons were excited by UTP, and the response was not blocked by PPADS. Although the percentage of cells responding to UTP is similar to that in the present study (83%, Table 2), an important difference is that, in the present study, only 20% of neurons responded to UTP in the presence of TTX. This suggests a role for P2Y2R and/or P2Y4R in regulating local afferents innervating the SON neurons, as well as mediating postsynaptic responses in a subset of neurons. Although Hiruma and Bourque (11) reported that their UTP response was TTX insensitive, only three neurons were tested in the presence of TTX.

Experiments designed to evaluate the source of the Ca2+ rise induced by ATP utilized established strategies, i.e., Ca2+-free medium to evaluate influx, thapsigargin to evaluate release from intracellular stores, and cadmium to evaluate influx through voltage-sensitive Ca2+ channels. As anticipated, P2XR activation induced Ca2+ influx through both voltage-sensitive Ca2+ channels and P2XR, but perhaps surprising was the finding that P2YR activation not only induced Ca2+ release from thapsigargin-sensitive stores (~80%), but also induced Ca2+ influx (the remaining 20% of the response). Possible mechanisms inducing this release include the following. 1) Activation of P2XRs by these agonists. This is not likely to be the explanation, since ATP still induced a small increase in [Ca2+]i in thapsigargin-pretreated explants exposed to PPADS or iso-PPADS (Fig. 4, B and D). 2) IP3 stimulation of Ca2+ influx. P2YR activation of Ca2+ influx was reported previously in oviduct mucosal cells (1). In these cells, IP3 receptor activation was required for the Ca2+ influx, and IP3 receptors were localized to the plasma membrane, leading to the hypothesis that plasma membrane IP3 receptor activation may directly trigger Ca2+ influx. IP3-induced Ca2+ influx has also been reported in Xenopus oocytes (7), but, to our knowledge, remains to be demonstrated in neurons. 3) Protein kinase C-induced Ca2+ influx. Other Gq/11-coupled receptors (e.g., muscarinic receptors) alter Ca2+ influx by activation of "receptor-operated Ca2+-channels" in neurons (17). Although the molecular mechanism mediating this phenomenon remains to be elucidated, the transient receptor potential vanilloid (TRPV) family of nonselective ion channels is a potential candidate. In SON, the stretch-inactivated cation channels involved in osmoreception, a major physiological regulator of VP secretion, are nonselective cation channels (20), and recent evidence suggests that these channels may be a variant of the TRPV1 channel (18). The stretch-inactivated cation channels in SON have previously been shown to be modulated by excitatory peptides, such as angiotensin, neurotensin, and cholecystokinin (4). All of these peptides activate Gq/11-coupled receptors (12, 14, 16, 26), and a Gq/ll-coupled P2YR has been shown to activate TRPV1 receptors in Neuro 2a cells (15). Thus, although further experiments are required to determine whether stretch-inactivated cation channels or TRPV1 channels contribute to the Ca2+ response to ATP in SON neurons, it is an intriguing possibility to pursue. If ATP can modulate channels involved in osmoreception via activation of P2YRs, it may elucidate the mechanism underlying the well-recognized modulation of osmotic regulation of VP secretion by hemodynamic stimuli (8, 24).


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Neurological Disorders and Stroke Grant R01-NS27975 to C. Sladek.


   ACKNOWLEDGMENTS
 
We thank Drs. Anne-Laure Perraud and Carsten Schmitz at National Jewish Medical and Research Center for use of PTI fluorometer and technical expertise.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Song, Dept. of Physiology and Biophysics, Univ. of Colorado at Denver and Health Sciences Center, P.O. Box 6511, Mail Stop 8307, Aurora, CO 80045 (e-mail: zhilin.song{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 REFERENCES
 

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