Roles of Purinergic P2X Receptors as Pacemaking Channels and ...

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Molecular Endocrinology 20(6):1423–1436 Copyright © 2006 by The Endocrine Society doi: 10.1210/me.2005-0508

Roles of Purinergic P2X Receptors as Pacemaking Channels and Modulators of Calcium-Mobilizing Pathway in Pituitary Gonadotrophs Hana Zemkova, Ales Balik, Yonghua Jiang, Karla Kretschmannova, and Stanko S. Stojilkovic Section on Cellular Signaling (H.Z., Y.J., K.K., S.S.S.), Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510; and Department of Cellular and Molecular Neuroendocrinology (H.Z., A.B.), Institute of Physiology, Academy of Sciences of the Czech Republic, Prague 142 20, Czech Republic Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but their biophysical properties and roles in spontaneous and receptor-controlled electrical activity have not been characterized. Here we focused on extracellular ATP actions in gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the Ca2ⴙ-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, nondesensitizing, and slow deactivating currents. In contrast, ATP induced nonoscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not Ca2ⴙ-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents

were consistent with the expression of P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations and LH release in an extracellular Ca2ⴙ-dependent manner. These inositol 1,4,5-triphosphate-dependent oscillations were facilitated, slowed, or stopped, depending of ATP concentration, the time of its application, and the level of Ca2ⴙ content in intracellular stores. These results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion. (Molecular Endocrinology 20: 1423–1436, 2006)

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tosolic domains. The ectodomain contains ATP binding site(s), both transmembrane domains contribute to the pore formation, and the intracellular domains participate in the control of permeability of the pore (3). A prolonged ATP application leads to a decrease in the conductivity of some P2XR subtypes, and this process is termed desensitization of receptors, whereas removal of agonist leads to deactivation of receptors (4). The rates of P2XR activation, desensitization, and deactivation are receptor specific. The efficacy of ATP and its analogs to activate channels is also receptor specific, as well as the blockade of ATP actions by antagonists, including pyridoxal 5-phosphate 6-azophenyl-2⬘,4⬘-disulphonic acid (PPADS), suramin, KN62, and reactive blue-2 (RB2) (2, 3). P2XRs are widely distributed in the brain, where mediate, presynaptically facilitate, and postsynaptically modulate fast synaptic transmission (5–9). In contrast to other ligand-gated receptor-channels, P2XR are also expressed in peripheral tissues where they participate in physiological processes as diverse as muscle contraction, secretion, blood clotting, and bone resorption (3). The mRNA transcripts for several P2XRs were also identified in anterior pituitary cells from adult rats, including the full-size P2X2R, termed P2X2aR (10) and

TP IS RELEASED by cells in a regulated manner (1) and can act as an extracellular messenger through activation of ligand-gated P2X receptor-channels (P2XRs) and G protein-coupled P2Y receptors (P2YRs) (2). P2XRs use the energy of ATP binding to initiate a depolarizing flux of cations through the pore of channels, which in turns activates voltage-gated Ca2⫹ influx. P2XR subunits are the products of seven genes and form homomeric and heteromeric channels. The functional channels are likely trimeric, and each subunit possesses a large ectodomain, two transmembrane domains, and amino and carboxy cyFirst Published Online March 16, 2006 Abbreviations: ATP␥S, Adenosine-5⬘-O-(3-thiotriphosphate); BzATP, 3⬘-O-(4-benzoyl)benzoyl-ATP; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-triphosphate; ␣␤-meATP, ␣␤-methylene-ATP; 2 Me-S-ATP, 2-methylthio-ATP; PPADS, pyridoxal 5-phosphate 6-azophenyl-2⬘,4⬘-disulphonic acid; P2XR, purinergic P2 receptor-channels; P2YR, G proteincoupled purinergic P2 receptors; RB2, reactive blue 2; SK channels, small calcium-activated K⫹ channels; Vm, membrane potential. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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the spliced form, termed P2X2bR (11), as well as P2X3R, P2X4R, and P2X7R (12). Functional expression of these receptors was documented using single cell Ca2⫹ measurements (12–14). Pituitary cells also express Ca2⫹-mobilizing P2YRs, which characterization was done using inositol phosphate measurements in mixed anterior pituitary cells (15) and single cell Ca2⫹ measurements in identified pituitary cell types (16–19). Furthermore, in dispersed pituitary cells, we observed an elevation in ATP release during application of GnRH (13), and others showed stimulatory effects of calcium ionophore A23187 on ATP release (17). Extracellularly added ATP is rapidly degraded by pituitary ectonucleotidase eNTPDase 1–3 (20). This enzymatic cascade generates ADP, the primary agonist for some P2YRs, and adenosine, the common agonist for purinergic P1 subtypes of receptors (21). Anterior pituitary cells express three subtypes of these receptors, A1, A2A, and A2B, and their activation leads to modulation of voltage-gated Ca2⫹ influx (22, 23). Although all necessary elements of purinergic signaling pathway are identified in anterior pituitary cells, the physiological relevance of ATP as an extracellular messenger is still not well established. As in a majority of other tissues expressing P2Rs, the cell type(s) responsible for ATP release, the nature of this process, the bulk and localized intercellular ATP concentrations, and the rates of ATP degradation have not been characterized in intact pituitary tissue. In addition, the biophysical properties of P2XRs expressed in pituitary cells have not been characterized, although such information are also critical for understanding the potential physiological roles of these channels in spontaneous and receptor-controlled electrical activity. In that respect, single cell Ca2⫹ measurements provided only a limited tool for characterization of native P2XRs in pituitary cells. These limits are discussed in details in Ref. (24). For example, recombinant P2X1R and P2X3R activate and desensitize in a millisecond-tosecond time scale (25, 26), which is insufficient time to generate global high amplitude Ca2⫹ signals. Pituitary cells also express voltage-gated Ca2⫹ channels (27), and Ca2⫹ influx through these channels and the handling of intracellular Ca2⫹ by cells influence the rates of ATP-induced Ca2⫹ signal desensitization and deactivation (28). Here we focus on biophysical characterization of P2Rs in gonadotrophs using single cell membrane potential (Vm) and current measurements. These cells are easy to identify in mixed subpopulations of dispersed anterior pituitary cells because of their specific expression of Ca2⫹-mobilizing GnRH receptors (29, 30). The other unique feature of these cells that is helpful in their identification is the oscillatory nature of inositol 1,4,5-triphosphate (InsP3)-dependent Ca2⫹ release from endoplasmic reticulum (ER), which in patch-clamp experiments can be monitored as periodic hyperpolarizing waves and baseline current oscillations mediated by Ca2⫹-activated and apaminsensitive K⫹ (SK) channels (31, 32). In all other

Zemkova et al. • Characterization of P2XRs in Gonadotrophs

secretory pituitary cells, Ca2⫹ mobilization occurs in a nonoscillatory manner. Our results indicate that identified gonadotrophs exclusively express P2X2Rs, and that these channels have capacity to contribute to the control of pacemaking activity and InsP3-dependent Ca2⫹ release mechanism.

RESULTS Electrophysiological Characterization of P2XR in Adult Gonadotrophs Embryonic, neonatal, and adult gonadotrophs express functional Gq/11 protein-coupled GnRH receptors and their stimulation leads to the generation of InsP3 and oscillatory Ca2⫹ release from intracellular stores (31– 33). Activation of other Ca2⫹-mobilizing receptors, including endothelin-A and pituitary adenylate cyclaseactivating polypeptide, as well as the injection of InsP3, also trigger periodic release of Ca2⫹ from ER (29). In our experiments, we used GnRH to identify gonadotrophs in all preparations. Figure 1A illustrates a typical profile of GnRH-induced SK current oscillations in voltage-clamped cells from adult animals. Similar oscillations were observed in gonadotrophs from embryonic and neonatal animals (data not shown). In none of gonadotrophs studied, application of ATP mimicked the action of GnRH on oscillatory SK current, clearly indicating that Ca2⫹-mobilizing P2YRs are not expressed in these cells. However, practically all gonadotrophs (46 of 47 GnRH-responsive cells tested) from adult animals responded to ATP application with the generation of an inward and nonoscillatory current (Fig. 1B), indicating that these cells express functional P2XRs. The peak amplitude of P2X current was dependent on ATP concentration (Fig. 1C), with an estimated EC50 of 13.6 ⫾ 3.8 ␮M (n ⫽ 5). The rate of current desensitization was also dependent on ATP concentration; Fig. 1B illustrates the rates of P2X current desensitization in the same cell stimulated with 10, 30, and 100 ␮M ATP for 60 sec. In contrast, the rate of P2X current deactivation (less than 0.3 sec) was independent of ATP concentration (Fig. 1, B and C). In response to supramaximal (100 ␮M) ATP concentration, the peak amplitudes of P2X currents varied between 10 pA and 500 pA, with the mean values of about 80 pA. In continuous presence of 100 ␮M ATP, receptors desensitized with a rate of about 16 sec (Fig. 2A). The resensitization of ATP-induced current occurred mono-exponentially, with a time constant of about 250 sec (Fig. 2B). The actions of ATP were mimicked by several agonists, with relative potency in order: 2Me-S-ATP⬎ATP ⫽ ATP␥S⬎BzATP. On the other hand, application of 100 ␮M ␣␤-meATP was ineffective. Such biophysical and pharmacological properties of P2X currents in gonadotrophs resemble the currents generated by

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Pacemaking Activity of P2XRs in Gonadotrophs

Fig. 1. Comparison of GnRH- and ATP-induced Currents in Identified Gonadotrophs A, GnRH-induced outward oscillatory SK current recorded using nystatin-perforated patch-clamp techniques in a cell clamped at ⫺40 mV. B, Dose-dependent effects of ATP on the rate of receptor desensitization and deactivation. C, Dose-dependent effects of ATP on peak current response. If not otherwise indicated, the records in this and following figures are done in gonadotrophs from adult animals. Horizontal bars above traces indicate the duration of GnRH and ATP application. HP, Holding potential.

recombinant P2X2R and/or P2X4R (3, 24). To clarify this issue, in further experiments we used suramin, PPADS, and RB2. P2X2R but not P2X4R are inhibited by suramin and PPADS (3), whereas among P2XRs RB2 only inhibits P2X2R (2). In pituitary gonadotrophs, ATP-induced current was blocked by suramin (Fig. 2C) and PPADS (data not shown). RB2 also inhibited ATP-induced current in gonadotrophs (Fig. 2D), confirming that these cells express exclusively P2X2 receptors.

In spontaneously active gonadotrophs, ATP increased frequency of action potential firing in a dose-dependent manner (Fig. 3A). At high (50 ␮M) concentrations, ATP-induced the bursting type of electrical activity (Fig. 3B). In quiescent cells, ATP initiated the firing of action potentials (Fig. 3E). The pacemaking activity of ATP was mimicked by BzATP and ATP␥S (Fig. 3, B and D), but not by ␣␤-meATP (Fig. 3C). In both quiescent and spontaneously active cells, the washout of agonists was followed by immediate abolition of depolarizing effects (Figs. 3 and 4A), which parallels with a rapid decay of current after washout of ATP (Figs. 1 and 2). Transient afterhyperpolarization observed at higher ATP doses inhibited electrical activity of spontaneously firing cells for 10–30 sec (Fig. 3, A and B). Consistent with the role of P2X2Rs in ATP-induced electrical activity in gonadotrophs, application of suramin (Fig. 3E) and PPADS (data not shown) abolished the stimulatory effects of ATP on pacemaking. These compounds applied alone did not affect baseline potential (Fig. 3E) and spontaneous firing of action potentials (data not shown). In contrast, application of RB2 induced rapid hyperpolarization of cell membrane (Fig. 3F) due to activation of potassium channels, which is consistent with findings in other cell types (34). Also, in RB2-treated cells, ATP was unable to depolarize plasma membrane (Fig. 3F). To study effects of pacemaking activity of ATP on LH secretion, we attached pituitary cells on beads, loaded cells into the chamber, and perifused them at a rate of about 1 ml/min at 37 C. Samples were collected every 4 sec and analyzed for their LH content. As shown in Fig. 4B, basal LH release is low, at the level of detection by RIA. This is consistent with published data showing the lack of action potential-secretion coupling in gonadotrophs, in contrast to somatotrophs, lactotrophs (35), and GnRH neurons (36). Application of ATP was associated with a relatively rapid activation of exocytosis, which reached the peak amplitude within 20 sec, followed by a gradual decay in secretion, with a time constant of about 25 sec (Fig. 4B, left panel). At the end of 150 sec ATP application, LH secretion was slightly above the basal level. The washout effect of ATP on LH secretion was studied in cells stimulated with 50 ␮M ATP for only 10 sec. In such treated cells, the decay of secretion was faster comparing to the rate decay of secretion in the presence of ATP (Fig. 4B, right panel). When compared with activation and deactivation phases of current, the time scale for rising and decay of exocytosis were longer, but the ratios in duration of onset and offset phases were highly comparable in both processes (about 1: 1.5). Furthermore, the time scales for rates of current and secretion desensitization during the continuous ATP application were comparable. These results suggest that the pacemaking activity stimulated by ATP leads to transient establishment of action potential-secretion coupling in gonadotrophs.

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Fig. 2. Identification of P2XR Subtype Expressed in Identified Gonadotrophs A, The rate of P2XR current desensitization during the sustained ATP application. Gray trace, Experimental record; black solid line, the monoexponential fitting curve for decay of current. Numbers below traces illustrate mean ⫾ SEM values for the peak current response and rates of receptor desensitization and deactivation in response to 100 ␮M ATP application from 35 experiments. The lack of rapid recovery form desensitization is illustrated by the size of current during the second ATP application. B, Prolonged recovery from desensitization during repetitive ATP stimulation of variable interpulse durations. C and D, Inhibition of ATP-induced current by suramin (C) and RB2 (D). Gray areas indicate duration of suramin and RB2 application. Experiments with RB2 were done at holding potential of ⫺80 mV to minimize effects of this compound on potassium current. The tailed outward current seen after cessation of ATP application (B and C) is due to transient activation of SK channels mediated by Ca2⫹ influx.

Comparison of the deactivation properties of GnRH and P2X2 receptors in gonadotrophs is shown in Fig. 4A. In contrast to P2XRs, GnRH receptor-induced current (Fig. 1A) and Vm (Fig. 4A) oscillations lasted for a prolonged period after removal of agonist, and the time for deactivation of these receptors increased with increase in GnRH concentrations. For example, the averaged washout time for 0.1 nM GnRH was 35 ⫾ 2 sec (n ⫽ 3), and for 1 nM GnRH was 90 ⫾ 23 sec (n ⫽ 5), compared with 1- to 2-sec washout time for ATPinduced depolarization. In parallel to P2XRs, the washout time for GnRH receptors was independent of duration of agonist application (Fig. 5, A–C). Such a slow deactivation of GnRH receptors reflected on the tail LH secretion in perifused cells. In cells stimulated with 1 nM GnRH for 5 min, the return of secretion to basal level after washout of agonists required almost 200 sec (Fig. 5D). Effects of ATP on GnRH-Induced Signaling Earlier studies indicated that the ER oscillator was spontaneously active in a small fraction of gonado-

trophs in static culture, generating low-frequency current/Ca2⫹ oscillations (37), whereas in the residual cells GnRH-dependent activation of phospholipase C was required for initiation of Ca2⫹ oscillations (38). Here we show that ATP-driven Ca2⫹ influx influenced both spontaneous and GnRH-induced Vm and current oscillations in the presence of GnRH and after its washout. As shown in Figs. 6–8, the actions of ATP were dependent on its concentration, the status of voltage-gated Ca2⫹ influx (voltage-clamped vs. current-clamped cells), and the level of depletion of the ER Ca2⫹ pool. ATP applied at high concentration after washout of GnRH immediately stopped Vm oscillations, followed by the increased firing frequency of action potentials (Fig. 6, A vs. B). Spontaneous SK-driven Vm oscillations were also stopped by ATP application (Fig. 6C). In the presence of GnRH, the actions of ATP were more complex. When applied during early phase of GnRH stimulation, ATP had three types of effects: it increased the frequency of action potential superimposed on depolarizing plateau without affecting the frequency of Vm oscillations (Fig. 6D), reduced the

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Fig. 3. The Pacemaking Role of P2XRs in Identified Gonadotrophs A, Dose-dependent effects of ATP on the frequency of action potentials and the level and duration of afterhyperpolarization in a spontaneously active cell. After 30 ␮M ATP application, the recovery of spontaneous firing required 30 sec. B, Comparison of the depolarizing effects of ATP and BzATP in a spontaneously active cell. C, The lack of ␣␤-meATP effects on firing of action potentials. D, Time-course of ATP␥S-induced depolarization in silent gonadotrophs. Horizontal bars above traces indicate the duration of agonist application. E, Attenuation of depolarizing effects of ATP by suramin. F, Hyperpolarizing effect of RB2 and the lack of depolarizing effect of ATP in RB2-treated cells.

frequency of Vm (Fig. 7A) and current (Fig. 7B) oscillations, or blocked GnRH-induced Vm (Fig. 7C) and current (Fig. 7D) oscillations in residual cells. Consistent with a rapid deactivation of P2XRs in gonadotrophs, all effects of ATP on ER oscillator in the presence of GnRH were transient. In cells bathed in Ca2⫹deficient medium, ATP generated inward current but was unable to influence GnRH-induced Vm and current oscillations (data not shown), indicating that Ca2⫹ influx is responsible for modulation of Ca2⫹-mobilizing pathway. The actions of extracellularly added ATP on GnRH-induced Vm and current oscillations were abolished in cells bathed in medium containing 100 ␮M suramin (data not shown). In cells clamped at ⫺60 mV to protect steady voltage-gated Ca2⫹ influx, the amplitude of Ca2⫹

and SK current oscillations decreased progressively during the prolonged GnRH application (39) due to depletion of intracellular Ca2⫹ pools (40). Figure 8 illustrates the rate of decrease in the peak amplitude of SK current oscillations. In these cells, ATP applied in 5–30 ␮M concentrations was able to partially recover the amplitude of GnRH-stimulated current oscillations without affecting the oscillatory nature of Ca2⫹ release from ER (Fig. 8, A–C). Furthermore, there was a rapid and transient recovery of the amplitude of spikes after washout of GnRH (Fig. 8, A and B), but the periodic ATP application in the absence of GnRH was not sufficient to keep the ER oscillator operative (Fig. 8C). ATP also increased the frequency of action potential superimposed on depolarizing plateau during the sustained GnRH appli-

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Fig. 4. Washout Effects of ATP on Vm and LH Release A, Comparison of the washout time for ATP- and GnRHinduced electrical activity. The extended time scales in A, bottom panels, illustrate the rapid washout effect of ATP on pacemaking. Horizontal bars above traces indicate the duration of GnRH and ATP application. B, Effects of ATP application and removal on LH secretion in perifused pituitary cells. Notice the difference in the time scales for left and right panels. Gray areas indicate the duration of ATP application. Horizontal bars above traces indicate the duration of agonist application.

cation, the pattern of which was highly comparable to that shown in Fig. 6D. These results suggest the importance of Ca2⫹ influx through the pore of P2XRs and by depolarization-driven activation of voltage-gated Ca2⫹ channels for sustain Ca2⫹ oscillations in GnRH-stimulated cells. Expression and Biophysical Characterization of P2XR in Neonatal Pituitary Cells The expression of mRNA transcripts for P2XRs in rat anterior pituitary cells from adult animals and GH3 immortalized pituitary cells was previously characterized (12). Here we show that transcripts for P2XRs were also present in anterior pituitary tissue from newborn and 7-d-old animals (Fig. 9A). Using specific rat P2XR primers, we identified transcripts for P2X1, P2X2, P2X3, P2X4, and P2X7 receptors in both groups. Functional P2X receptors were also identified in embryonic and neonatal pituitary cells. The resting potential in neonatal gonadotrophs was about ⫺50 mV, and a

Fig. 5. Washout Effects of GnRH on Vm Oscillations in Single Gonadotrophs and LH Release in Perifused Pituitary Cells A–C, Independence of deactivation time of duration of GnRH application. Gray areas indicate duration of GnRH application. D, The tail secretion after removal of GnRH in perifused pituitary cells. Cells were stimulates with GnRH for 5 min, and the record shows the end of GnRH pulse (indicated by gray area) and the washout period.

majority of cells did not fire action potentials spontaneously. However, application of ATP led to a rapid and transient depolarization of cells and generation of action potentials (Fig. 9B). ATP-induced depolarization of cells was abolished in the presence of suramin (Fig. 9C) and PPADS (data not shown). Due to limitation in total number of cells harvested from neonatal anterior pituitaries, we did not study LH secretion by rapid perifusion. In cells in static culture, ATP significantly enhanced GnRH-induced LH release (Fig. 9D), con-

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trophs from adult animals, removal of agonist was followed by a rapid deactivation of receptors. ATPinduced current was abolished in cells perfused with RB2 (Fig. 10B) the specific blocker of P2X2Rs. PPADS (Fig. 10C) and suramin (Fig. 10D) also inhibited ATPinduced current in neonatal gonadotrophs. Recombinant P2X2Rs, but not other subtypes of these receptors, are inhibited by elevated extracellular calcium concentrations (3). When neonatal gonadotrophs were perfused with 5 mM Ca2⫹-containing medium, ATPinduced current response was reduced in a reversible manner (Fig. 10E). Finally, P2X4R, but not P2X2R, P2X3R, and P2X7R, are sensitive to ivermectin (41). In cells expressing recombinant P2X4R, ivermectin increases the maximum current amplitude, decreases the EC50 for ATP, and greatly prolongs the deactivation of current after ATP removal (41, 42), whereas in neonatal gonadotrophs, this compound was ineffective (data not shown). Taken together, these results indicate that functional P2X2Rs were expressed in pituitary gonadotrophs during embryonic and adult life and contribute to the purinergic signaling in anterior pituitary cells.

DISCUSSION

Fig. 6. Effects of ATP on Spontaneous and GnRH-induced Vm Oscillations A, Typical washout profile of Vm oscillations in cells exposed to GnRH for 5 sec. B, Termination of GnRH washout effect on Vm oscillations by ATP. Notice the increased frequency of action potentials after removal of ATP. C, Termination of spontaneous Vm oscillations by ATP. D, ATP-induced increase in the firing frequency of action potentials in cells with activated Vm oscillations by GnRH. Horizontal bars below traces indicate the duration of GnRH and ATP pulses.

firming the amplifying role of ATP on sustained GnRHstimulated Ca2⫹ signaling and secretion. P2X current was identified in both embryonic (Fig. 10A, top panels) and neonatal pituitary cells (Fig. 10A, bottom panels). ATP-induced current was observed in 20 of 22 cells. The peak amplitude of current varied between 10 pA and 220 pA, with mean value of 67 ⫾ 12 pA. The rates of current desensitization during the prolong agonist application varied among the gonadotrophs (Fig. 10A, top vs. bottom panel), with the mean desensitization time constant of 18.6 ⫾ 4.5 sec (n ⫽ 10) in response to 100 ␮M ATP. As with gonado-

We believe this is the first study on biophysical and electrophysiological characterization of native P2XRs in an anterior pituitary cell type. Our results indicate that practically all GnRH-responsive cells express functional P2XRs, which generated a depolarizing and nonoscillatory current. In none of the gonadotrophs examined, ATP triggered oscillatory SK current in the absence of GnRH stimulation, further indicating that Ca2⫹-mobilizing P2YRs are not expressed in this particular cell type. The biophysical properties of P2X currents (kinetics of activation, deactivation, desensitization, and resensitization) in gonadotrophs were comparable with those observed in cells expressing recombinant P2X2R and/or P2X4R. Specifically, the rates of desensitization of native P2XRs in gonadotrophs were faster than desensitization of recombinant P2X2a and P2X7R, slower than desensitization of recombinant P2X1R and P2X3R, and highly comparable to the rates of desensitization decays of recombinant P2X4R, P2X2bR, and heteromeric P2X2a/P2X2bR (24, 28). In gonadotrophs, ␣␤-meATP, a partial agonist for P2X4R (41), triggered current in none of cells examined, a finding more consistent with the conclusion that native channels in these cells are P2X2abR. Experiments with suramin, PPADS, RB2, elevated extracellular Ca2⫹, and ivermectin confirmed this conclusion. In accordance with our findings, earlier studies showed the robust expression of mRNA transcripts for the full-size and spliced forms of P2X2Rs in anterior pituitary cells (10, 11). Here we also show that the P2X2R transcripts and functional channels were present in embryonic and 7-d-old rats. Others showed

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Fig. 7. Effects of ATP Application on Early GnRH-Induced Vm (Top Panels) and Current (Bottom Panels) Oscillations A and B, ATP-mediated decrease in the frequency of Vm and current oscillations induced by 1 nM GnRH. C and D, Transient and repetitive blockade of GnRH-induced Vm and current oscillations by extracellular ATP. Horizontal bars above traces indicate the duration of GnRH and ATP application.

that P2X2R subtype is also expressed in the brain from embryonic 10-d rats (43), as well as in GnRH-secreting neurons from embryonic 35- to 37-d rhesus monkeys (36). In general, P2X2Rs are rapidly activated in response to ATP application and the pores of these channels are permeable to monovalent and bivalent cations, including Ca2⫹, which results in depolarization of Vm (44). Pituitary gonadotrophs express numerous voltageand ligand-gated channels, including L-type Ca2⫹ channels (27), and exhibit spontaneous firing of action potentials, associated with small amplitude of Ca2⫹ transients (45). Here we show that the ATP-induced depolarizing current initiated the firing of action potentials in quiescent cells and increased the frequency of spiking in spontaneously active cells. At saturating ATP concentrations, this current was sufficient to generate the plateau-bursting type of electrical activity. Earlier studies showed that ATP generates global high amplitude Ca2⫹ signals (12–14). Such ATP-induced Ca2⫹ influx was of sufficient amplitude to trigger exocytosis. Thus, P2X2Rs in gonadotrophs have the potential to operate as pacemaking channels. Our results also indicate that P2X2Rs in gonadotrophs can modulate GnRH-induced electrical activity, Ca2⫹ signaling, and LH secretion, and that effects of ATP on GnRH-induced currents and Vm oscillations were strictly dependent on extracellular Ca2⫹ influx, a finding consistent with the mechanism of oscillatory Ca2⫹ release in gonadotrophs. GnRH receptors trigger baseline Ca2⫹ oscillations by increasing the intracellular InsP3 levels (38), whereas cytosolic Ca2⫹ exhibits biphasic effects on InsP3-dependent oscillations,

stimulatory at low levels and inhibitory at high levels (37). The isolated InsP3-gated receptor-channels are also activated by an increase in InsP3 concentration and display a bell-shaped curve for dependence on Ca2⫹ (46, 47). In a theoretical model of pituitary gonadotrophs, such a dual control of InsP3 channel gating combined with the activity of ER Ca2⫹-ATPase, which pumps Ca2⫹ back to ER, is sufficient to generate oscillatory Ca2⫹ release from ER (48). Thus, activation of an additional Ca2⫹ influx pathway mediated by P2X2R in cells with operative ER oscillator should effectively change the coagonist actions of InsP3 and Ca2⫹ in controlling the gating of InsP3 receptor channels. The results further suggest that the impact of ATPinduced Ca2⫹ influx on the coagonist actions of InsP3 and Ca2⫹ was determined by the status of intracellular Ca2⫹ pool. During the brief exposure to GnRH, the intracellular Ca2⫹ pool is only partially affected and Ca2⫹ concentrations at peak Ca2⫹ spikes are high (40). ATP effectively stopped the sustained InsP3-dependent Vm oscillations after removal of GnRH, while increasing the frequency of action potentials. In the presence of GnRH, ATP was able to reduce the frequency of InsP3-dependent Vm and SK current oscillations, or to transiently stop the oscillator, but only in a fraction of cells and during the early phase of GnRH application. Therefore, we suggest that under these conditions facilitation of extracellular Ca2⫹ influx by ATP enhanced the inhibitory effects of Ca2⫹ on InsP3 receptor-channels, leading to a decrease in the frequency of spiking or a transient blockade of the ER oscillator activity.

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Fig. 8. Refilling of Intracellular Calcium Stores by P2XR-Driven Calcium Influx A–C, Time-course of ATP actions on the amplitude of sustained GnRH-induced current oscillations in gonadotrophs clamped at ⫺60 mV. Cells were continuously stimulated with GnRH and ATP was applied in a repetitive manner in the presence and absence of GnRH. Notice an increase in the amplitude of oscillatory current during ATP application and immediately after its washout.

On the other hand, during the sustained activation of GnRH receptors, intracellular Ca2⫹ pools are gradually depleted (40) due to activity of the plasma membrane Ca2⫹-ATPase, Na⫹/Ca2⫹ exchange system, and the uptake of intracellular Ca2⫹ by mitochondria (49–51). This in turn leads to a progressive decrease in the peak amplitude of Ca2⫹ spikes (39, 45). Furthermore, when the ER pool is depleted, the ER Ca2⫹-ATPase plays a critical role in keeping oscillations alive, whereas the negative effect of Ca2⫹ is limited (45). Here we show that in voltage-clamped cells during prolonged GnRH stimulation, ATP increased the amplitude of InsP3dependent oscillations and transiently supported the

operation of ER oscillator after removal of GnRH. In current-clamped cells, ATP increased the frequency of action potential firing in a majority of cells. Basically, in cells with depleted intracellular Ca2⫹ pool ATP helps to sustain such oscillations by facilitating Ca2⫹ influx through the pore of P2XR, as well as by facilitating the pacemaking activity during the depolarizing phases of GnRH-induced oscillations. Such a stimulatory effects of ATP-induced Ca2⫹ influx on ER oscillations provides the potential rationale for the amplification of LH release during the sustained GnRH stimulation. Our results also indicated that deactivation of GnRH receptors required tens of seconds and was depen-

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Fig. 9. Expression and Role of P2XR in Embryonic and Neonatal Pituitary Cells A, Detection of five P2XR mRNA transcripts in anterior pituitary tissues from newborn and 7-d-old animals. B, Depolarizing effect of ATP in an identified neonatal gonadotroph. C, Attenuation of depolarizing effects of ATP by suramin in neonatal gonadotrophs. D, Amplification of GnRHinduced LH release by ATP in neonatal pituitary cells in static culture. LH release is expressed as percentage of maximum stimulated with 100 nM GnRH (100%). Asterisks indicate significant differences between the pairs.

dent on agonist concentration. It is highly unlikely that GnRH washout time reflects the dissociation time for GnRH receptor-agonist complex. The delay in deactivation of GnRH receptor signaling rather indicates the slow silencing of phospholipase C activity and gradual degradation of intracellular InsP3. Because the mam-

Zemkova et al. • Characterization of P2XRs in Gonadotrophs

Fig. 10. Characterization of P2X Current in Embryonic and Neonatal Pituitary Cells A, ATP-induced inward currents in identified gonadotrophs from 20-d-old embryos (upper traces) and 5-d-old rats (bottom traces). B–E, Inhibition of ATP-induced inward current by RB2 (B), PPADS (C), suramin (D), and extracellular calcium (E) in neonatal gonadotrophs. Gray areas indicate duration of application of P2XR blockers, and horizontal bars indicate duration of ATP application. Experiments were performed in cells at holding potential of ⫺60 mV, except for experiments with RB2, which were done at holding potential of ⫺80 mV.

malian GnRH receptors are nondesensitizing (30), these data also suggest that the periodic in vivo GnRH release and slow deactivation of GnRH receptor-dependent signaling in gonadotrophs provides an economical system to extend the duration of LH pulses after termination of GnRH release from hypothalamus

Zemkova et al. • Characterization of P2XRs in Gonadotrophs

and the washout/degradation of intrapituitary GnRH (52). Potentiation of GnRH-induced secretion by pituitary-derived ATP might also represent a positive feedback mechanism by which hormone secretion is stimulated without interfering with the pulsatile GnRH release. In contrast to GnRH, the ATP actions on gonadotroph functions could be controlled by two mechanisms: desensitization of P2XR and deactivation of channels. At saturating ATP concentrations, P2X current in these cells desensitized within a few minutes, whereas removal of ATP induced a rapid deactivation of P2X2 current (in a millisecond time scale), cessation of depolarization, and a transient hyperpolarization. This first process could provide a mechanism for protection of cells from harmful effects of extreme Ca2⫹ influx during excessive ATP release, and the second process could lead to synchronization of electrical activity in intact tissue. In vivo, ectonucleotidase eNTPDases 1–3, which are expressed in pituitary cells (20), provide an effective pathway for control of ATP extracellular actions. At the present time, it is difficult to assess the physiological relevance of pituitary purinergic signaling system in control of gonadotroph functions in the absence of in situ data about ATP release, the balance between de novo ATP release and its degradation, and the impacts on such equilibrium on pacemaking activity and GnRH-induced oscillations. We know that ATP is released by dispersed pituitary cells (13, 20), and that the bulk ATP concentrations in perifusion medium measured by ATP bioluminescent assay kit are below the threshold levels required for activation of P2X2 receptors in gonadotrophs. However, ATP could also be released by other cell types in response to other stimuli. Furthermore, our assay and other assays based on firefly luciferase (1, 20) do not allow detection of the rapidly released ATP close to the surface of the plasma membrane. Other studies suggested that ATP concentrations in the proximity of plasma membrane surface are 10- to 20-fold higher than the ATP concentrations measured in the bulk solution (53). In addition, experiments with the plasma membranetargeted luciferase indicated that extracellular ATP concentrations are in the range of 100–200 ␮M (54), which is more than sufficient to activate all types of P2XRs and P2YRs. Because our biophysical measurements were done in dispersed single cells seated at low density, perfused at a rate that is sufficient to remove the micromolar to milimolar ATP concentrations around the cells in a millisecond time scale, the physiological relevance of localized ATP release could not be tested. In summary, these investigations indicate that extracellular ATP operating via P2X2Rs has the capacity to control the pacemaking activity, voltage-gated Ca2⫹ influx, and basal LH release in gonadotrophs. Because other anterior pituitary cells also express P2Rs (12, 14, 16), it is reasonable to speculate that ATP may serve as a synchronizer of spontaneous elec-

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trical activity through the control of afterhyperpolarization periods. ATP may also initiate intercellular Ca2⫹ waves, as has been observed in other cell types (55). Finally, Ca2⫹ influx through the pore of P2XRs and the associated voltage-gated Ca2⫹ influx could play important roles in modulation of GnRH-induced and InsP3-mediated oscillatory Ca2⫹ release. Such an action of ATP could provide a mechanism for amplification of GnRH effects on calcium signaling and secretion. Further experiments should clarify to which extend these capacities of P2XRs are used in vivo.

MATERIALS AND METHODS Chemicals 2Me-S-ATP was purchased from Calbiochem EMD Bioscience, Inc. (La Jolla, CA) and pluronic F-127 was obtained from Molecular Probes (Eugene, OR). GnRH was obtained from Peninsula Laboratories (Belmont, CA), PPADS from Tocris Cookson Inc. (Ellisville, MO), suramin from EMB Biosciences (San Diego, CA), and reactive blue-2 from Axxora (Alexis) (San Diego, CA). If not otherwise specified, all other chemicals were from Sigma (St. Louis, MO). Cell Cultures Experiments were performed on anterior pituitary cells from normal female Sprague Dawley rats at different developmental stages, obtained from Taconic Farms (Germantown, NY) and the Animal Facility at the Institute of Physiology, Prague. Pituitary cells from adult animals were dispersed as described previously (12) and cultured in medium 199 containing Earle’s salts, sodium bicarbonate, 10% heat-inactivated horse serum, and penicillin (100 U/ml) and streptomycin (100 ␮g/ml) (all from Invitrogen, Carlsbad, CA). Neonatal pituitary cells were harvested from 4- to 7-d-old rats as described in Ref. 56. In some experiments, pituitary from embryos (embryonic d 20) were also used. Experiments were approved by the National Institute of Child Health and Human Development Animal Care and Use Committee, and the Animal Care and Use Committee of the Academy of Sciences of the Czech Republic. RT-PCR Expression Analysis of P2X Receptor Subtypes Total RNA was extracted from anterior lobe of the pituitaries from Sprague-Dawley rats using RNeasy Mini Kit (QIAGEN, Valencia, CA). Material was collected from two groups of pups (newborn and 7 d old). During extraction, total RNA was treated with ribonuclease-free deoxyribonuclease I (QIAGEN) for 20 min at room temperature. Quantity of extracted RNA was measured on BioPhotometer (Eppendorf, Hamburg, Germany). First-strand cDNA was synthesized from 2 ␮g of total RNA using 500 ng random primers (Invitrogen) and 200 U Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) in total volume of 25 ␮l. Control reactions in the absence of reverse transcriptase were also carried out. Completed reactions were terminated by heating at 70 C for 10 min. Synthesized cDNA/control was diluted to final volume of 50 ␮l. For RT-PCR analysis, we used the P2XR sequencespecific primers as described previously (57). PCR was performed with 2 ␮l of diluted cDNA using the sense and antisense primers (400 nM each) in total volume of 50 ␮l, containing 200 ␮M each of four deoxynucleotide triphosphates, 1.5 mM MgCl2 and one unit of Taq DNA polymerase

1434 Mol Endocrinol, June 2006, 20(6):1423–1436

1.1 (Top-Bio, Prague, Czech Republic). The conditions of a manual hot-start PCR were as follows: 92 C for 5 sec, pause step to add polymerase, subsequently 92 C for 30 sec, 62 C for 45 sec, and 72 C for 2 min for 37 cycles, with a final extension at 72 C for 10 min. For negative controls, PCR was conducted using first strand cDNA samples without reverse transcriptase. PCR samples were size-fractionated in 1% agarose gel and visualized with ethidium bromide staining. Electrophysiological Recordings Membrane voltage potential and whole-cell currents were measured using nystatin-perforated patch-clamp technique at room temperature. Cells were continuously perfused with an extracellular solution containing (in mM): 150 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.3 with NaOH. Patch pipettes were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL) and polished by heat to a tip resistance of 5–7 M⍀. Pipette solution contained (in mM): 70 K-aspartate, 70 KCl, 3 MgCl2, and 10 HEPES; pH was adjusted to 7.2 with KOH. Before measurement, nystatin and dispersing agent pluronic F-127 were added to the intracellular solution from stock solutions to obtain final concentrations of 250 ␮g/ml and 500 ␮g/ml, respectively. Recordings were done 10 min after seal formation. The plasma membrane potential was held at ⫺60 mV, if not otherwise stated. Current-clamp and voltage-clamp recordings were performed using Axopatch 200B amplifier (Axon Instruments, Union City, CA). Data were captured and stored using the pClamp 8 software packages in conjunction with the Digidata 1322A A/D converter (Axon Instruments). No series resistance compensation was used and the corrections of Vm for the Donnan potential and liquid junction potential were ignored. Solutions were delivered to recording chamber by a gravity-driven microperfusion system (ALA Scientific Instruments, Westbury, NY). The application tip was routinely positioned at about 500 ␮m distance and about 50 ␮m above the recorded cell. Less than 200 msec were required for exchange of solutions around the patched cells. LH Measurements Hormone secretion was monitored using cell column perifusion chambers and static cultures. In perifusion experiments 1.5 ⫻ 107 cells were incubated with preswollen cytodex-1 beads in 60-mm Petri dishes for 20 h. The beads were then transferred to 0.5 ml chambers and perifused with Hanks’ M199 containing 25 mM HEPES, 0.1% BSA, and penicillin (100 U/ml)/streptomycin (100 ␮g/ml) for 2.5 h at a flow rate of 0.25 ml/min at 37 C to establish stable basal secretion. Fractions were collected in 4-sec intervals and immediately assayed for LH contents. For measurements of LH release by neonatal gonadotrophs, cells in static cultures (1.5 ⫻ 105/well) were washed and then stimulated with increasing concentrations of GnRH in the presence or absence of 10 ␮M ATP for 4 h. LH content was measured by RIA, using rat LH antiserum (rabbit) (NIDDKanti-rLH-S-11), rat LH standard (NIDDK-rLH-RP-3), and antigen for iodination (NIDDK-rLH-I-10) provided by the National Pituitary Agency and Dr. A. F. Parlow (HarborUCLA Medical Center, Torrance, CA). Tracer was iodinated by 125I[Na] in the presence of iodogen and separated on Sephadex column. The inter- and intraassay coefficients of variation were 9.2% and 2.1%, respectively; the sensitivity of the assay was 20 pg. Data Analysis Concentration-response data were fitted by a sigmoid curve using Sigma Plot 6.1 software. This program was also used

Zemkova et al. • Characterization of P2XRs in Gonadotrophs

for evaluating statistical significances. The ATP-induced current amplitudes were measured using the program CLAMPFIT 8 (Axon Instruments). The rate of desensitization and the kinetics of current decay evoked by washout of agonists (deactivation) were fitted by a single exponential function y ⫽ A exp(⫺t/␶), where A is the amplitude and ␶ is a time constant. The rate of recovery from desensitization was fitted using equation I ⫽ Imax [1 ⫺ exp(⫺t/␶rec)], where I is the observed peak current response, Imax is the maximum peak current recovery, t is the washing time, and ␶rec is the recovery time constant. All numerical values in the text are reported as mean ⫾ SEM, and significant differences, with P ⬍ 0.05 were determined by Student’s t test.

Acknowledgments Received December 12, 2005. Accepted March 7, 2006. Address all correspondence and requests for reprints to: Dr. Stanko Stojilkovic, Section on Cellular Signaling, Endocrinology and Reproduction Research Branch/National Institute of Child Health and Human Development/National Institutes of Health, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: [email protected]. This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health, and the Academy of Sciences of the Czech Republic Grants (AVOZ50110509, A5011408, and A5011103). H.Z., A.B., Y.J., K.K., and S.S.S. have nothing to declare.

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