Possible Role of 5'-Adenosine Triphosphate in Synchronization of Ca2+ Oscillations in Primate Luteinizing Hormone-Releasing Hormone Neurons

Ei Terasawa, Kim L. Keen, Richard L. Grendell and Thaddeus G. Golos

Wisconsin National Primate Research Center (E.T., K.L.K., R.L.G., T.G.G.), and Departments of Pediatrics (E.T.) and Obstetrics and Gynecology (T.G.G.), University of Wisconsin, Madison, Wisconsin 53715-1299

Address all correspondence and requests for reprints to: Ei Terasawa, Wisconsin Regional Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: terasawa{at}primate.wisc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LHRH neurons derived from the olfactory placode region of monkey embryos exhibit spontaneous intracellular Ca2+ ([Ca2+]i) oscillations that synchronize among LHRH neurons and nonneuronal cells at a frequency similar to pulsatile LHRH release. To understand the mechanism of intercellular communication between LHRH neurons and nonneuronal cells, which leads to synchronization, we examined the possible role of ATP. 1) ATP, not ADP or AMP, stimulated both LHRH release and [Ca2+]i concentration, whereas the ATP-induced [Ca2+]i response was abolished by infusion of apyrase, which hydrolyzes ATP; 2) the ATP-induced [Ca2+]i response occurred in normal (but not low) extracellular Ca2+ and was blocked by the voltage-dependent L-type Ca2+ channel blocker, nifedipine; 3) pharmacological experiments with purinergic receptor agonists and antagonists indicated that the ATP-induced [Ca2+]i response in LHRH neurons was mediated through P2X, but not P2Y, receptors; 4) cloning and sequencing studies suggested that P2X2 and P2X4 transcripts were present in olfactory placode cultures; and 5) P2X2 receptors and P2X4 were expressed in LHRH neurons. The results suggest that ATP may play a role in intercellular communication when LHRH neurons synchronize, and raise the possibility that nonneuronal cells, such as glia, may be a crucial component of the in vivo LHRH neurosecretory system.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN PRIMATES, LHRH is released in pulses at hourly intervals, and this pulsatility is essential for the maintenance of normal reproductive function (1). However, the mechanism of pulsatile LHRH release is still unclear. Progress studying molecular and cellular mechanisms of pulsatility in LHRH neurons is hampered by their paucity in number and scattered distribution pattern in the hypothalamus, where they do not form a particular nucleus, as seen with magnocellular neuroendocrine neurons.

Accordingly, several years ago we established an in vitro culture system for LHRH neurons derived from the olfactory placode region of the rhesus monkey (2). In this in vitro system, LHRH neurons release the decapetide in a pulsatile manner at intervals of approximately 1 h even though LHRH neurons were of embryonic origin, as long as they were cultured more than 2 wk (3). Moreover, we have found that LHRH neurons also exhibit synchronizations of intracellular Ca2+ ([Ca2+]i) oscillations at hourly intervals (4) and synchronization of [Ca2+]i oscillations is propagated as a Ca2+ wave over LHRH neurons and nonneuronal cells (5). Although the question of whether synchronization of [Ca2+]i is associated with LHRH neurosecretion remains to be answered, these observations led us to propose a hypothesis that LHRH neurons and nonneuronal cells are functionally integrated and that nonneuronal cells are involved in synchronizing the activity of the LHRH neurosecretory network (5). If nonneuronal cells, such as glia in the hypothalamus, play a similar role in synchronizing isolated LHRH neurons in the hypothalamus, this would provide an indirect coupling mechanism to facilitate pulsatile release of LHRH neurons in vivo.

As a first step to understand intercellular communication between LHRH neurons and nonneuronal cells in our culture system, in the present study we investigated the possible role of ATP as a chemical substrate for intercellular communications. This was based on recent reports showing that extracellular ATP plays a significant role in intercellular communications between neurons and glia (6, 7, 8). Extracellular purines (adenosine, ADP, and ATP) and pyrimidines [uridine diphosphate and uridine triphosphate (UTP)] are important signaling molecules that mediate diverse biological effects via cell-surface receptors, which include P1 receptors for adenosine and P2 receptors for ATP, ADP, UTP, and uridine diphosphate (9). P2 receptors are further divided into two families, ionotropic P2X receptors (ligand-gated ion channels) and metabotropic P2Y receptors (G protein-coupled receptors) (9, 10, 11). Here we report that LHRH neurons express P2X2 and P2X4 receptor subunits and ATP alters activity of LHRH neurons. We hypothesize that ATP is a chemical substrate mediating intercellular signals to propagate calcium waves through P2X receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of ATP on LHRH Release in LHRH Neurons
Infusion of ATP at 50 µM over olfactory cultures stimulated LHRH release (Fig. 1AGo), whereas vehicle infusion did not change the release pattern (Fig. 1BGo). ATP at 1 µM did not induce any significant effects. Neither ADP nor AMP at 50 µM resulted in significant effects (data not shown).



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Fig. 1. ATP at 50 µM Stimulates LHRH Release in Cultured LHRH Neurons

ATP dissolved in artificial cerebrospinal fluid (A) or vehicle (artificial cerebrospinal fluid; B) was infused for 20 min, whereas perifusate samples were collected at 10-min intervals. LHRH levels in perifusates were assessed by RIA. Note that a significant increase in LHRH release (P < 0.05) was observed during the ATP infusion.

 
Effects of ATP on [Ca2+]i Release in LHRH Neurons
ATP at 0.1–100 µM resulted in [Ca2+]i increases in LHRH neurons in a dose-responsive manner (Fig. 2Go, B and C). ATP-induced [Ca2+]i increases appeared as a single large peak or a single peak followed by multiple peaks during the descending phase (Fig. 2BGo). When the ATP dose was low (0.1 µM), either the frequency of [Ca2+]i oscillations increased (Fig. 2BGo), or the amplitude increased slightly, or no response was observed (data not shown). When the ATP dose was high (10 to 100 µM), a large [Ca2+]i increase with a baseline shift during the ascending phase followed by multiple [Ca2+]i pulses during the descending phase was observed (Fig. 2Go, A and B). The calculated ED50 of ATP effects on [Ca2+]i response was 2 µM (Fig. 2CGo). The [Ca2+]i response to high ATP (100 µM) was smaller than its response to high K+ (56 mM), although the response to ATP varied among individual cells. In general, 56 mM K+ induced a single peak response with a larger amplitude (1.5- to 2.5-fold) and a longer duration (approximately 2-fold), in which multiple [Ca2+]i pulses during the descending phase were absent. Interestingly, ATP at 0.1–1 µM for 5 min occasionally resulted in a cluster of synchronizations with intervals at 2.5–10 min (Fig. 3Go). Repeated applications of ATP over 1 µM caused a small desensitization (data not shown). In contrast, ADP, AMP (Fig. 2AGo), UTP, and adenosine at 10 µM failed to increase [Ca2+]i consistently in LHRH neurons (data not shown). The absence of a response to adenosine excludes the possible involvement of P1 receptors and the absence of a response to UTP and ADP excludes the possible involvement of P2Y receptors. Thus, stimulatory effects of ATP on [Ca2+]i concentrations in LHRH neurons appear to be mediated by P2X receptors.



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Fig. 2. ATP Increases [Ca2+]i Concentrations in LHRH Neurons in a Dose-Responsive Manner

Two examples of the effects of ATP, ADP, and AMP for 5 min application on [Ca2+]i concentrations from one culture are shown in panel A. ATP, but not ADP and AMP, is effective to increase [Ca2+]i concentrations. The effects of ATP on [Ca2+]i concentrations are dose dependent, as seen in the two examples from one culture with ATP at 0.1, 1, 10, and 100 µM (B) and the dose response curve (ATP at 10 nM to 1 mM) of the mean {Delta}[Ca2+]i concentrations (C). There was considerable difference in the response to ATP among individual LHRH neurons. The number of cultures in each dose (C) was five to six, except for the 1 mM dose that was from two cultures. The ED50 was 2 µM. The equation for the curve fit was Y = –0.58139X3 – 9.9807X2 – 49.426X – 53.984, and the correlation was R2 = 0.992.

 


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Fig. 3. ATP-Induced Repeated Synchronization of [Ca2+]i Oscillations

ATP at a dose of 0.1 µM was infused for 5 min, as indicated by a black bar, and the ATP-induced synchronizations lasted for 20–25 min. The example is shown from nine LHRH neurons in one culture.

 
ATP also stimulated [Ca2+]i increases in nonneuronal cells, a group of cells with uniform shape with relatively large cell nuclei. However, in contrast to the response of LHRH neurons 1) these cells often responded to ADP and AMP, but not adenosine, with a small amplitude, 2) ATP at 1–100 µM resulted in consistent responses, but a low dose (0.1 µM) was not effective, and the [Ca2+]i response at 1 µM in nonneuronal cells was smaller than that in LHRH neurons, and 3) nonneuronal cells were readily desensitized by ATP at 1 µM, i.e. the second exposure to ATP dramatically reduced the response. Fibroblasts did not respond to ATP. Nonneuronal cells failed to respond to a high K+ challenge.

Infusion of apyrase (20–80 U/ml), which hydrolyzes ATP blocked the ATP-induced [Ca2+]i increase and suppressed spontaneous [Ca2+]i pulses (Fig. 4Go) indicating that not only exogenous ATP causes the [Ca2+]i increase, but endogenous ATP may also contribute to the generation of [Ca2+]i pulses.



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Fig. 4. The Hydrolysis of ATP (1 µM) by Apyrase (20 U/ml) Blocked the ATP-Induced Increase in [Ca2+]i Concentrations in LHRH Neurons

A, ATP was infused over cultures for 5 min and apyrase was infused for 11 min, starting 3 min before ATP and ending 3 min after ATP. Ayrase (20 U/ml) suppressed spontaneous [Ca2+]i oscillations in LHRH neurons (B). Two representative examples in each experiment are shown.

 
Response of LHRH Neurons to P2X Receptor Agonists and Antagonists
To determine which P2X receptor subunits are involved in ATP action in LHRH neurons, we examined various agonists and antagonists for P2X receptors. Seven P2X receptor subunits (P2X1 to P2X7) have been described (9, 10, 11, 12). First, adenosine 5'-O-3-[{gamma}-thio]-triphosphate (ATP-{gamma}S), a P2X2/P2X3 agonist, induced an [Ca2+]i increase, although the response was not as large as the ATP-induced response (Figs. 5Go and 6AGo). Second, 2-methylthioadenosine 5'-triphosphate (2MeSATP), an agonist for P2X2, P2X4, and P2X5, induced an [Ca2+]i increase and the response to 2MeSATP was as large as or larger than ATP (Fig. 6AGo). Third, {alpha},ß-methylene-adenosine-5'-triphosphate ({alpha},ß-ATP), a P2X1, P2X4, and P2X5 agonist, induced an [Ca2+]i increase, but the response to {alpha},ß-ATP was significantly smaller than that induced by ATP-{gamma}S as well as ATP (Fig. 6AGo). Fourth, LHRH neurons did not respond to diadenosine pentaphosphate (AP5A), a P2X3 agonist. Fifth, 3'-benzoylbenzoyl-ATP (BzATP) at 10–500 µM, which is a 10-fold more potent agonist than ATP for P2X7 (10), did not result in any [Ca2+]i increase (Fig. 6AGo). Sixth, the ATP-induced [Ca2+]i increase was blocked by reactive blue 2 (RB2), a specific P2X2 receptor blocker, as well as by suramin and pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), which were nonspecific P2X2 blockers (Fig. 6BGo). These results from pharmacological experiments suggest that the effects of ATP in LHRH neurons are mediated by P2X receptors, which appear to have the properties of P2X2, P2X4 and P2X5 subunits.



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Fig. 5. The Effects of ATP-{gamma}S on [Ca2+]i Concentrations in LHRH Neurons

ATP-{gamma}S also stimulates [Ca2+]i concentrations in a dose-responsive manner but the responses were not as large as those seen with ATP. ATP-{gamma}S was infused for 5 min. Two examples from one culture are shown.

 


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Fig. 6. The Effects of Various Agonists (A) and Antagonists (B) of ATP on [Ca2+]i Concentrations in LHRH Neurons

A, {Delta}[Ca2+]i (mean ± SEM) obtained with exposure to ATP and its agonists at the dose of 10 µM were compared. B, ATP-induced increase in [Ca2+]i concentrations was blocked by antagonists. The dose of ATP, PPADS, suramin, and RB2, were 10, 10, 100, and 10 µM, respectively. Note that in panel A 2MeSATP was as effective as ATP, whereas the responses induced by ATP-{gamma}S and {alpha},ß-ATP were smaller than those induced by ATP. AP5A and BzATP did not induce any effect. **, Means significantly different from ATP and 2MeSATP at P < 0.001. B, PPADS, suramin, and RB2 themselves did not resulted in any effect, whereas they blocked the effect of ATP. **, Means significantly different from ATP at P < 0.001; {dagger}, means significantly different from the respective antagonist alone at P < 0.01. This series of experiments lead to the conclusion that the ATP-induced increase in [Ca2+]i concentrations was mediated by P2X2 and/or P2X4 receptors (see text). The mean data were derived from a total of three to 11 cultures per group containing a total of 32–242 cells.

 
The Requirement of Extracellular Ca2+ (Ca2+e) for the ATP-Induced [Ca2+]i Increase
The ATP-induced [Ca2+]i increase requires the presence of [Ca2+]e because the effects of ATP were blocked in low [Ca2+]e (Fig. 7AGo). Moreover, L-type Ca2+ channels are involved in the action of ATP, as the ATP-induced [Ca2+]i increase was modified by the presence of nifedipine, an L-type Ca2+ channel blocker. Nifedipine at either 10 or 50 µM blocked the [Ca2+]i increase induced by ATP at 1 µM (Fig. 7BGo). Interestingly, the suppression of the ATP-induced [Ca2+]i increase by nifedipine was dependent on the relative concentration with ATP. That is, the [Ca2+]i elevation induced by a dose of ATP, for example 1 µM, was reduced by increasing doses of nifedipine (Fig. 7CGo). Alternatively, the suppressive effects of nifedipine, for example at 10 µM, were overcome when the dose of ATP was increased (data not shown). These observations suggest that ATP may not only depolarize the membrane through inward Na+ current but also result in Ca2+ influx. The N-type Ca2+ channel blocker, {omega}-conotoxin GVIA did not interfere with the ATP-induced [Ca2+]i response (data not shown).



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Fig. 7. The Presence of Normal [Ca2+]e Concentrations Is Necessary for the Effect of ATP

A, Effects of ATP at 10 µM were examined in a normal [Ca2+]e solution (1.25 mM) or low [Ca2+]e (20 nM) solution. The L-type Ca2+ channel blocker, nifedipine, blocks the ATP-induced increase in [Ca2+]i concentrations (B). B, Effects of ATP (1 µM) were examined with or without the presence of nifedipine at 10 and 50 µM. The reduction of the nifedipine doses (0.1–1 µM) from 10 µM was less effective in suppressing the [Ca2+]i increase by 1 µM ATP (C). Representative examples in three single cells were obtained from three different cultures.

 
Molecular Identification of P2X Receptor Subunits in the Monkey Brain and Placode Cultures
Based on the pharmacological experiments, P2X2, P2X4, and P2X5 subunits appear to play a role in ATP effects in LHRH neurons. Using primers derived from human cDNA sequences, we examined whether P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 mRNAs were present in olfactory placode cultures and the preoptic area of the rhesus monkey. P2X6 mRNA was not examined because of the unavailability of P2X6 cDNA sequence in humans. RT-PCR produced cDNA products identified as P2X2 and P2X4 in both olfactory placode cultures and the preoptic area (Fig. 8Go). Interestingly, P2X4 cDNAs obtained from the olfactory placode cultures and the preoptic area differed by three nucleotides. In contrast, P2X7 was not detected in placode cultures, despite the fact that it was found in the preoptic area (Fig. 8Go). P2X1, P2X3, and P2X5 cDNAs were not detected in either the olfactory placode cultures or the preoptic area (P2X1 and P2X5 data not shown). P2X2 cDNAs obtained from olfactory placode cultures, and the preoptic area reveal several variants of the molecule, some of which putatively code for truncated forms of the protein, i.e. the transcripts putatively coded for proteins similar to P2X2b and a form of P2X2 encompassing both of the deletions found in P2X2c and P2X2 h. In total, we isolated nine variants of P2X2, one from the preoptic area and eight from the placode (data not shown). Because the detailed results of cloning and sequencing are beyond the scope of this manuscript, we will describe these observations in detail in a later publication.



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Fig. 8. RT-PCR Amplification of P2X2, P2X4, and P2X7 from the Preoptic Area (POA), and P2X2 and P2X4 from Olfactory Placode Cultures (PL)

P2X2 and P2X4 amplicons (around 1100 bp, indicated by asterisks) from both the preoptic area and placode, and the P2X7 amplicon (around 1700 bp, indicated by an asterisk) from the preoptic area were cloned, sequenced, and identified as their respective genes. All other amplicons from the P2X2, P2X3, P2X4, and P2X7 lanes in both tissues were cloned, sequenced, and shown to be unrelated to the P2X receptor family. LHRH (200 bp) and G3PDH (1000 bp) PCRs were used as positive controls for each tissue.

 
These results indicate that several types of P2X receptors are present in the monkey hypothalamus, that their cDNA sequences are similar but not identical with those found in humans, and that mRNAs for P2X2 and P2X4 receptor subunits are present in olfactory placode cultures.

Colocalization of P2X2 and P2X4 Receptors in LHRH Neurons
To identify P2X receptor subunit proteins in LHRH neurons, we double immunostained cultures with antibodies for LHRH and one of the antibodies for P2X1, P2X2, P2X3, P2X4, or P2X7. Analysis with a confocal microscope indicated that LHRH neurons (Fig. 9Go, A and D) were double stained for P2X2 (Fig. 9Go, B and C) and P2X4 (data not shown), but not for P2X1, P2X3, and P2X7 (Fig. 9Go, E and F). Similar results were obtained from LHRH neurons in hypothalamic tissue sections. Interestingly, a subset of LHRH neurons, rather than all LHRH neurons, were double stained for either P2X2 or P2X4. In contrast, nonneuronal cells, but not LHRH neurons, in cultures were P2Y1 and P2Y2 immunopositive (data not shown).



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Fig. 9. The Presence of P2X2 in Cultured LHRH Neurons

LHRH-positive cells shown in green (A), are also P2X2 positive in red (B), whereas LHRH-positive cells in green (D) are P2X7 negative (E). Colocalization of P2X2 and LHRH (C) is shown in yellow. P2X7 is not colocalized in LHRH neurons (F). Note that a large red blot in the right corner (B and C) is an artifact in the tissue culture, and not all LHRH neurons are P2X2 immunopositive. Scale bar, 50 µm for all photomicrographs.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we found that ATP stimulates LHRH release, agonists and antagonists of P2X receptors modify the activity of LHRH neurons, and LHRH neurons express P2X2 and P2X4 subunits.

Because truly specific agonists and antagonists for P2X receptor subunits are not available and the specificity of agonists and antagonists for P2X receptor subunits described in the literature are inconsistent (this is partly due to a relative paucity of P2X receptor studies), we needed to conduct an extensive series of pharmacological studies. For example, the absence of a response to BzATP was puzzling, because BzATP is an agonist for all subunits (10). Nonetheless, in the early stages of this study we could predict the involvement of P2X2 in ATP action and speculate the presence of P2X4 in LHRH neurons.

Cloning and sequencing studies suggested that olfactory placode cultures contained P2X2 and P2X4 mRNA. In fact, RNA extracts from the olfactory placode cultures and preoptic area expressed several forms of P2X2 mRNA. Moreover, we found that LHRH neurons express P2X2 and P2X4 proteins. Molecular cloning of P2X receptor subunits indicates that a subunit is composed of a 379- to 472-amino acid protein, in which the N and C terminals are in the cytoplasm connected by two-transmembrane-spanning segments and a large extracellular loop (9, 12). Because a single two-transmembrane subunit alone cannot form an ion channel, it is speculated that a receptor is assembled with multiple subunits (11, 12). The presence of P2X2 and P2X4 subunits indicate that P2X2 and P2X4 subunits may form homomeric P2X2 and P2X4 receptors or P2X2/P2X4 heteromeric receptors in LHRH neurons. We cannot, however, exclude the possibility that LHRH neurons have other subunits. The failure to immunostain other subunit proteins in LHRH neurons could be due to the low expression of subunits, and the failure to detect mRNA for other subunits in olfactory placode culture could be due to the design of the primers that we used in this study. The electrophysiological properties of LHRH neurons in response to ATP and P2X agonists and antagonists would further clarify the composition of P2X subunits in future experiments. Nonetheless, it is important to note that LHRH neurons are equipped with P2X receptors, through which an elevation of ATP can alter activity of LHRH neurons.

P2X receptors are ATP-gated ion channels, which mediate rapid and selective permeability to cations, Na+, K+, and Ca2+. In the present study, we have found that the ATP-induced increase in [Ca2+]i requires the presence of [Ca2+]e and this ATP effect is blocked by a high dose of the voltage-sensitive L-type Ca2+ channel blocker nifedipine. If the nifedipine dose is lower or the ATP dose is higher, the blockage by nifedipine is partial. A similar [Ca2+]e dependency and partial blockage of the ATP-induced Ca2+ signaling by nifedipine has also been reported in rat pituitary cells (13). Based on our observation we can propose two possible mechanisms, which are not mutually exclusive. First, an increase in extracellular ATP leads to an activation of P2X receptors allowing the entry of nonspecific cations, such as Na+, resulting in slight depolarization of the membrane. Subsequently, the ATP-induced slight membrane depolarization leads to an opening of the voltage-sensitive Ca2+ channels (the voltage-sensitive L-type and perhaps T-type Ca2+ channels), followed by Ca2+ influx, resulting in further depolarization of the membrane as well as the Ca2+-induced Ca2+ release from intracellular stores, and ultimately leading to an induction of action potentials and neurosecretion. Second, ATP may have selectively allowed the Ca2+ entry through P2X2/P2X4 receptors, because it has been shown that the ATP-induced activation of P2X2/P2X4 receptors have a high permeability to Ca2+ (10) and nifedipine may have indirectly blocked Ca2+ entry. In fact, the observation that suppressive effects of nifedipine were relative to the dose of ATP supports the second mechanism. Mechanisms of the sequence of events need to be investigated with electrophysiological approaches.

It has been reported that GT1 cells do not express P2X receptors (13). This difference in primate LHRH neurons and GT1 cells is unclear. However, there are also many other differences these cell types: LHRH neurons in the present study are primary cells derived from the embryonic olfactory placode region of rhesus monkeys, whereas GT1 cells are of mouse origin, carrying transcripts of rat LHRH as well as tumor genes.

Many types of anterior pituitary cells express P2Y and P2X receptors and release ATP (14). Rat gonadotropes express P2X receptors, and ATP stimulates an increase in [Ca2+]i concentrations in gonadotropes and LH release (15). Moreover, LHRH challenge to gonadotropes induces ATP release (15). Although we did not measure ATP in our cultures, it is likely that ATP is released when [Ca2+]i concentrations increase in LHRH neurons. The observation that apyrase suppressed spontaneous [Ca2+]i oscillations indicates that the presence of ATP, presumably released from cells in a culture, influences an increase in [Ca2+]i concentrations.

In previous studies, we have found that LHRH neurons exhibit periodical synchronizations of [Ca2+]i oscillations (4, 5). A signal for synchronization originates from LHRH neurons and is propagated across nonneuronal cells as a calcium wave (5). GT1–1 cells, not GT1–7 cells, also exhibit periodical synchronizations of [Ca2+]i oscillations, which can be seen as Ca2+ waves across a population of cells (16, 17). It has been shown that ATP is an intercellular messenger in cortical (18) and retinal (7) astrocytes, in which calcium waves are propagated by ATP. Neurons release ATP with other neurotransmitters, such as GABA, glycine, and glutamate (19). Glia also release ATP in response to mechanical or electrical stimulation (8, 20, 21) and influence activity of neighboring neurons by modulating presynaptic and postsynaptic transmissions using ATP (7, 8, 22). Both neurons and glial cells express purinergic receptors (6). Moreover, bidirectional communication between neurons and glia at the synapse has been extensively reported (11, 23, 24). Therefore, it is possible that LHRH neurons and nonneuronal cells use ATP as an intercellular messenger. In fact, ATP infusion at a small dose occasionally resulted in frequent synchronization of [Ca2+]i oscillations with short intervals at 2.5–10 min. As described previously, periodical synchronizations of [Ca2+]i oscillations in our cultures normally occur at 50- to 60-min intervals (4, 5), this is a highly significant phenomenon. These results suggest that ATP appears to play a role in synchronization of [Ca2+]i oscillations, although the mechanism is yet to be investigated.

In the present study, we have observed that ATP stimulates LHRH release in olfactory placode cultures. We have also found that ATP induces an increase in [Ca2+]i levels in both LHRH neurons and nonneuronal cells. LHRH neurons express P2X2 and P2X4 receptors and nonneuronal cells express P2Y1 and P2Y2 receptors. Thus, LHRH neurons and nonneuronal cells appear to communicate using extracellular ATP. Because the synchronization of [Ca2+]i oscillations requires the presence of LHRH neurons (5), ATP may be released from LHRH neurons to initiate calcium waves. ATP released from LHRH neurons would alter the activity of nonneuronal cells, which in turn would modulate activity of LHRH neurons. Although our cultures do not contain glial cells in most cases, and although the identity of nonneuronal cells in our cultures has yet to be thoroughly defined, the observation in this study provides some insights into events in the hypothalamus. LHRH neurons are widely scattered in the hypothalamus and preoptic area (25), and there are few synaptic interactions or gap-junctional interactions among them (26). If the mechanism observed in this study occurs among LHRH neurons and glia in the hypothalamus, scattered LHRH neurons can communicate with each other with the aid of glia. Collectively, we propose the hypothesis that glia are a part of the LHRH pulse-generating system participating in pulsatile LHRH release in vivo. This hypothesis needs to be examined further in the future.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals, Tissue Preparations, and Cultures
A total of 14 fetuses at embryonic d 35–37 obtained from time-mated pregnant female rhesus monkeys (Macaca mulatta) were used in this study. Fetuses were delivered by Cesarean section under isoflurane anesthesia. The procedures for time-mated pregnancy and calculation of fetal age were described previously (2). For the cloning of P2X receptors, the brain tissues from two adult female rhesus monkeys were obtained through the Tissue Distribution Program of the Wisconsin National Primate Research Center. All experiments presented in this manuscript were performed following the standards established by the Animal Welfare Act and the document entitled "Principles for Use of Animals and Guide for the Care and Use of Laboratory Animals." The protocol for this study was reviewed and approved by the Animal Care and Use Committee, University of Wisconsin.

Tissues for cultures were prepared as described previously (2). Briefly, the olfactory placode and ventral LHRH neuron migratory pathway region were dissected out into chilled plating medium (imidazole-buffered L15 medium; Invitrogen Life Technologies, Grand Island, NY), cut into very small pieces (<0.5 mm3), and two to three pieces of tissue were plated onto round (25 mm diameter) glass coverslips with engraved grids (No. 2, Bellco Glass, Inc., Vineland, NJ). Coverslips were coated with a layer of dried rat-tail collagen. Cultures on coverslips were maintained in 35-mm tissue culture dishes (Corning, Corning, NY) containing growth medium (Medium 199, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 0.6% glucose, 50 µg/ml gentamicin, and L-glutamine (0.1 µg/ml) and incubated at 37 C with 1.5% CO2 and 98.5% air (2). On d 3–4 in culture, cells were exposed to antimitotic agent 5-fluoro-5-deoxyuridine 30–40 µM for 3 d. Medium was replaced every 3–4 d at the beginning of cultures and every 1–2 d after the cultures were established. All experiments were conducted on cells cultured for 2–4 wk.

Recording of [Ca2+]i Concentrations
The method for measurement of [Ca2+]i was the same as that described previously (4). [Ca2+]i was measured by calculating the ratio of the fluorescence intensity ({Delta}F/F0) of the Ca2+ indicator dye, fura-2 AM (Texas Fluorescence Labs, Austin, TX; cells loaded with 18 µm for 30 min at 37 C) with cells excited at 340 and 380 nm (133-msec delay). Light emitted at 510 nm was captured by a video camera (Hamamatsu Photonics, Hamamatsu, Japan) at 10-sec intervals. Cultures were perfused continuously with oxygenated (95% O2 and 5% CO2) medium (Medium 199, Sigma; 50 µl/min) at room temperature for 100–200 min. A culture was viewed through a microscope with a x20 objective, and a 750 x 750-µm recording field that contained the appearance of LHRH neurons (2) was selected for data capture. LHRH neurons were identified readily during Ca2+ imaging according to their morphology (ovoid, highly fluorescent soma; one or more somatic processes) as well as their migratory pattern, and were generally easily distinguishable from nonneuronal cells. Nonneuronal cells were described in four categories based on their appearance: placode cells, epithelial-like cells with a round, uniform shape, fibroblasts, and unidentifiable cells. Measurements of fluorescence in individual cells were made by delimiting the borders of the cell body on a video image and measuring pixel intensity within the borders of the digitized fluorescence image. Fluorescence data were captured and analyzed using commercially available software (Metafluor, Universal Imaging Corp., West Chester, PA). The free [Ca2+]i concentration was estimated using the equation described by Grynkiewicz et al. (27) from the ratio image. The equation is [Ca2+]i = Kd{(R-Rmin)/(Rmax-R)}Sf2/ Sb2, where R is the ratio of the light emitted when the dye is excited by the two excitation wavelengths, Rmin and Rmax are the values of R at very low and high Ca2+ concentrations, respectively, Sf2 and Sb2 are intensities of free and bound fura-2 at 380 and 340, respectively, and Kd is the effective dissociation constant of fura-2 under these particular experimental conditions.

Effects of depolarizing agents on [Ca2+]i concentrations were tested as follows: K+ at 28 or 56 mM; the voltage sensitive L-type channel antagonist, nifedipine at 10 and 50 µM, and the voltage sensitive N-type Ca2+ channel antagonist {omega}-conotoxin GVIA at 1 µM. Effects of low [Ca2+]e concentrations were tested by reducing [Ca2+]e to 20 nM from normal 1.25 mM. In this case, salt balance was maintained by adjusting the Mg2+ concentration (1.32 mM). K+ challenge was routinely given at the end of experiments to confirm the presence of LHRH neurons from background cells.

The following purinergic receptor agonists and antagonists were tested: ATP, ADP, AMP, and UTP at 0.1, 1, 10, 50, and 100 µM; 2MeSATP, ATP-{gamma}S, {alpha},ß-ATP, AP5A at 0.1, 1, 10, and 100 µM; BzATP at 1, 10, 100, and 500 µM; PPADS at 1, 10, and 100 µM; suramin at 1, 10, and 100 µM; and RB2 at 10 and 100 µM. Similarly, the effect of apyrase, which hydrolyzes ATP, at 20–80 U/ml was examined. For each chemical or dose, at least three cultures and up to 11 cultures (typically four to six cultures) were examined. The dose response curve of ATP at doses between 0.1 and 100 µM was derived from five experiments, in which all doses were examined in one experiment, and the ATP data at doses of 0.01 µM and 1 mM were obtained from additional five and three experiments, respectively. Because of potential desensitization by any of these chemicals, the order of different chemical application to cells as well as the order of the doses of ATP and other ATP agonists and antagonists were randomized in each experiment. All chemicals except for 2MeSATP (Tocris, Cookson Inc., Ellisville, MO) were obtained from Sigma.

Immunocytochemistry
LHRH neurons were further identified by the procedures detailed previously with minor modifications (4). Briefly, once a recording experiment was complete, the imaged area was photographed and a coverslip grid reference was obtained to facilitate locating the cells after immunostaining. LHRH neurons were immunostained with a cocktail of antisera GF-6 and LR-1 (gifts from Dr. N. M. Sherwood, University of British Victoria, Victoria, Canada; 1:9,000 dilution and Dr. R. A. Benoit, University of Montreal, Montreal, Canada; 1:15000, respectively) and 3,3'-diaminobenzadine as the chromagen, as described previously (4, 5). In general, cultures were composed of LHRH-immunopositive neurons and nonneuronal cells, such as fibroblasts, epithelial cell marker-positive cells, and unidentified cells. LHRH-immunopositive neurons were distinguished from other types of neurons by their red-brown color staining and matched to the photographic and digitized fluorescence images that were acquired before immunostaining. Neurons immunonegative to LHRH were seldom seen, but if we found them they were excluded from analysis. In selected cultures, an attempt to identify the cell type of nonneuronal cells was made with immunostaining for epithelial cell-specific antigen (catalog no. E6011; Sigma, St. Louis, MO). Nonneuronal cells that participate in synchronization were often immunopositive for epithelial cell marker protein. In the present study, however, we retain the term "nonneuronal cells" because we did not identify the cell type of nonneuronal cells in all cultures.

To identify colocalization of P2X and P2Y receptor subunits with LHRH neurons, cultures were first immunostained for P2X1, P2X2, P2X3, P2X4, P2X5, P2X7, P2Y1, or P2Y2 (all polyclonal antisera made in goat from Santa Cruz Biotechnology, Santa Cruz, CA) with rhodamine and then exposed to the monoclonal antiserum for LHRH, HU4H (gift from Dr. Henryk Urbanski, Oregon National Primate Research Center; 1:800) (28) and labeled with fluorescein isothiocyanate. Double fluorescent immunostaining has been described previously (29) and analysis was performed using a confocal microscope at the Keck Biological Imaging Laboratory, University of Wisconsin-Madison.

Cell Perifusion and LHRH Assays
Two to 4 wk after the initiation of culture, the effect of ATP, ADP, AMP (all at 50 µM), or vehicle was examined by perifusion experiments. Two coverslips with cells were mounted in Sykes-Moore chambers and perifused with a modified Krebs-Ringer phosphate buffer containing 0.1% glucose (pH 7.4) and 58 µg/ml bacitracin under 95% O2 and 5% CO2 at 37 C and perifusates were collected at 32 µl/min in 10-min fractions using the ACUSYST (Endotronics, Minneapolis, MN) perifusion system, as described previously (3). ATP ADP, AMP, or vehicle was applied for 20 min. All samples were stored at –70 C until assay for LHRH. After the perifusion experiment, cells were fixed with 2% paraformaldehyde (pH 7.6) and immunostained for LHRH or other antibodies as described above.

LHRH concentrations in media samples were measured in duplicate samples of 150 µl each with RIA using antiserum R-1245 (a gift from Dr. T. Nett, Colorado State University, Fort Collins, CO) as described previously (30). Synthetic LHRH (Richelieu Laboratory, Inc., Montreal, Canada) was used for both the trace and the reference standard. The standard curve was constructed using the culture medium as a part of the assay buffer. The sensitivity of the assay at 95% binding was 0.05 pg/tube. Intra- and interassay coefficients of variation were 8.3% and 11.4%, respectively.

RT-PCR, Cloning, and Sequencing
To determine whether the rhesus monkey brain and olfactory placode region contain P2X mRNAs, total RNA from the preoptic area of two adult rhesus monkey brains and 20 olfactory placode cultures from six fetuses were isolated using RNA STAT-60 (Tel-Test, Friendswood, TX). Individual RNA samples were then analyzed for the presence of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 mRNAs using RT-PCR.

For each reverse transcription (RT) reaction, 1 µg of total RNA was brought up to a total volume of 20 µl using the GeneAmp RNA PCR Core Kit (Applied Biosystems, Branchburg, NJ). Oligo deoxythymidine16 primers were used in each reaction according to manufacturer’s specifications. After RT, 3- to 5-µl aliquots of each reaction were used in PCR. PCR primers designed to detect P2X mRNAs were based on human cDNA sequences and were synthesized by the University of Wisconsin Biotechnology Center. Each RT aliquot used for PCR was combined with 2.5 mM deoxynucleotide triphosphates (Amersham Biosciences, Princeton, NJ), 1x PCR Buffer II with MgCl, 1.25 U AmpliTaq (Applied Biosystems, Branchburg, NJ), and 12.5 pmol of either P2X, LHRH, or glyceraldehydes-3-phosphate dehydrogenase (G3PDH) primers in a final volume of 50 µl. LHRH and G3PDH primers were used as PCR-positive controls. Negative controls for PCR used aliquots of water mixed with each PCR/primer cocktail. PCR amplification conditions consisted of one cycle at 94 C for 1 min, 55 C for 30 sec, 72 C for 1.5 min; 35 cycles at 94 C for 30 sec, 55 C for 30 sec, 72 C for 1.5 min; and an incubation at 72 C for 7 min.

After PCR amplification, 20-µl aliquots of each reaction were fractionated on a 1% agarose TBE gel, and products were visualized using ethidium bromide staining. Target cDNA amplicons were isolated and purified with the Geneclean II kit (Bio101, La Jolla, CA), and subcloned into pcDNA2.1 Original TA cloning vector (Invitrogen, Carlsbad, CA). Subcloned fragments were sequenced on an AB1 3700 DNA analyzer (University of Wisconsin Biotechnology Center) using the BigDye Terminator mix (PE Applied Biosystems, Foster City, CA).

PCR primer sequences and GenBank accession numbers referenced for their design are shown below.

P2X1
Upstream primer (5'-CCT CTT CGA GTA TGA CAC-3') and downstream primer (5'-GAT GTC CTC ATG TTC TCC-3') were derived from the alignment of 3 human P2X1 mRNA sequences (GenBank accession nos. NM_002558, AF020498, and U45448). Upstream and downstream primers start at positions 36 and 1179 within the coding region of these mRNAs.

P2X2
Upstream primer (5'-CTA CGA GAC GCC CAA GGT GAT CG-3') and downstream primer (5'-CAG AGT TGA GCC AAA CCT TTG G-3') were derived from the alignment of 10 human P2X2 mRNA sequences encompassing the splice variants P2X2A, P2X2B, P2X2C, P2X2D, and P2X2H (GenBank accession nos. AF109387, AF190822, AF260426, AF109388, AF190823, AF260427, AF190824, NM_016318, AF190825, and AF260428). The upstream primer is located in the coding region just 5' of the deletion of the P2X2H variant, and the downstream primer is located in the coding region just before the stop codon.

P2X3
Upstream primer (5'-GCA TAT CCG ACT TCT TCA CC-3') and downstream primer (5'-CTA GTG GCC TAT GGA GAA GG-3') were derived from the alignment of three human P2X3 mRNA sequences (GenBank accession nos. AB016608, NM_002559, and XM165609). Upstream and downstream primers start at positions 8 and 1175 within the coding region of these mRNAs.

P2X4
Upstream primer (5'-TCC TGT TCG AGT ACG ACA CG-3') and downstream primer (5'-TCA CTG GTC CAG CTC ACT AGC-3') were derived from the alignment of 3 human P2X4 nucleotide sequences (GenBank accession nos. AF191093, NM_002560, and U83993). Upstream and downstream primers start at positions 32 and 1147 within the coding region of their respective mRNA sequences.

P2X5
Upstream primer (5'-CTG TTC GAC TAC AAG ACC-3') and downstream primer (5'-AAT TCA CGT GCT CCT GTG-3') were derived from a human P2X5 mRNA sequence (GenBank accession no. NM_002561). Upstream and downstream primers start at positions 37 and 1255 within the coding region of this mRNA.

P2X7
Upstream primer (5'-AAG TCA CTC GGA TCC AGA GC-3') and downstream primer (5'-CAG TAA GGA CTC TTG AAG CC-3') were derived from a human P2X7 mRNA sequence (GenBank accession no. Y09561). Upstream and downstream primers start at positions 50 and 1768 within the coding region of this mRNA.

LHRH
Upstream primer (5'-ACT CCA GCC AGC ACT GGT CCT ATG G-3') and downstream primer (5'-ACC TGC CCA GTT TCC TCT TCA ATC A-3') were derived from a M. mulatta mRNA fragment (GenBank accession no. S75918). Upstream and downstream primers start at positions 11 and 193 of this mRNA fragment.

G3PDH
Upstream primer (5'-TGA AGG TCG GAG TCA ACG GAT TTG-3') and downstream primer (5'-CAT GTG GGC CAT GAG GTC CAC CAC-3') were derived from a human G3PDH mRNA sequence (GenBank accession no. BCO14085). Upstream and downstream primers start at positions 11 and 970 within the coding region of this mRNA.

Data Analysis
[Ca2+]i levels were processed on Excel spread sheets. Recorded cells were labeled as either LHRH neurons or non-LHRH cells, based on the criteria described above. The [Ca2+]i response to ATP agonists and antagonists were calculated in each cell as the difference between the base line level (mean of six [Ca2+]i levels before the challenge to the drug) and the peak [Ca2+]i value during the drug exposure. Group mean (±SEM) from all cells in a culture was calculated from individual data. The dose-response curve of ATP was derived from the [Ca2+]i response mean of three to six cultures, containing nine to 32 cells per culture. A synchronization of [Ca2+]i oscillations was defined to occur when the peak of [Ca2+]i increase within 20–60 sec at a higher proportion than average coincidence, using the calculation method described previously (5).

The effect of ATP on LHRH release was examined by comparing the data from vehicle using the analysis of variance repeated measure followed by post hoc analysis with Student-Newman-Keuls’ test. Similarly, the effects of ATP agonists and antagonists on the [Ca2+]i response were examined with the analysis of variance repeated measure followed by post hoc analysis with Student-Newman-Keuls’ test. Differences between groups were considered to be significant when P < 0.05.


    ACKNOWLEDGMENTS
 
We thank Rafael Connemara for his technical assistance.


    FOOTNOTES
 
This work is supported by National Institutes of Health Grants HD15433, HD11355, and 5P51RR000167.

First Published Online June 30, 2005

Abbreviations: AP5A, Diadenosine pentaphosphate; {alpha},ß-ATP, {alpha},ß-methylene-adenosine-5'-triphosphate; ATP-{gamma}S, adenosine 5'-O-3-[{gamma}-thio]-triphosphate; BzATP, 3'-benzoylbenzoyl-ATP; [Ca2+]e, extracellular Ca2+; [Ca2+]i, intracellular Ca2+; G3PDH, glyceraldehydes-3-phosphate dehydrogenase; 2MeSATP, 2-methylthioadenosine 5'-triphosphate; PPADS, pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid; RB2, reactive blue 2; RT, reverse transcription; UTP, uridine triphosphate.

Received for publication January 13, 2005. Accepted for publication June 23, 2005.


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