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.
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ABSTRACT |
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INTRODUCTION |
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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.
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RESULTS |
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Infusion of apyrase (2080 U/ml), which hydrolyzes ATP blocked the ATP-induced [Ca2+]i increase and suppressed spontaneous [Ca2+]i pulses (Fig. 4) 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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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. 9, A and D) were double stained for P2X2 (Fig. 9
, B and C) and P2X4 (data not shown), but not for P2X1, P2X3, and P2X7 (Fig. 9
, 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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DISCUSSION |
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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). GT11 cells, not GT17 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 2.510 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.
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MATERIALS AND METHODS |
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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 34 in culture, cells were exposed to antimitotic agent 5-fluoro-5-deoxyuridine 3040 µM for 3 d. Medium was replaced every 34 d at the beginning of cultures and every 12 d after the cultures were established. All experiments were conducted on cells cultured for 24 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 (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 100200 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 -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-S,
,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 2080 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 manufacturers 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 2060 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online June 30, 2005
Abbreviations: AP5A, Diadenosine pentaphosphate; ,ß-ATP,
,ß-methylene-adenosine-5'-triphosphate; ATP-
S, adenosine 5'-O-3-[
-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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REFERENCES |
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