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Coexposure of hypothalamo-neurohypophyseal system explants to ATP and phenylephrine [PE; an alpha1-adrenergic receptor (alpha1-AR) agonist] induces an extended elevation in vasopressin and oxytocin (VP/OT) release. New evidence is presented that this extended response is mediated by recruitment of desensitization-resistant ionotropic purinergic receptor subtypes (P2X-Rs): 1) Antagonists of the P2X2/3 and P2X7-Rs truncated the sustained VP/OT release induced by ATP+PE but did not alter the transient response to ATP alone. 2) The P2X2/3 and P2X7-R antagonists did not alter either ATP or ATP+PE-induced increases in [Ca(2+)](i). 3) P2X2/3 and P2X7-R agonists failed to elevate [Ca(2+)](i), while ATP-gamma-S, an agonist for P2X2-Rs increased [Ca(2+)](i) and induced a transient increase in VP/OT release. 4) A P2Y1-R antagonist did not prevent initiation of the synergistic, sustained stimulation of VP/OT release by ATP+PE but did reduce its duration. Thus, the desensitization-resistant P2X2/3 and P2X7-R subtypes are required for the sustained, synergistic hormone response to ATP+PE, while P2X2-Rs are responsible for the initial activation of Ca(2+)-influx by ATP and ATP stimulation of VP/OT release. Immunohistochemistry, coimmunoprecipitation, and Western blot analysis confirmed the presence of P2X2 and P2X3, P2X2/3, and P2X7-R protein, respectively in SON. These findings support the hypothesis that concurrent activation of P2X2-R and alpha1-AR induces calcium-driven recruitment of P2X2/3 and 7-Rs, allowing sustained activation of a homeostatic circuit. Recruitment of these receptors may provide sustained release of VP during dehydration and may be important for preventing hemorrhagic and septic shock.

The enteric nervous system (ENS) controls gut function. P2X receptors and nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that mediate fast synaptic excitation in the ENS. Close molecular coupling in enteric neuronal membranes contributes to a mutually inhibitory interaction between these receptors; this effect is called cross-inhibition. We studied the molecular mechanisms responsible for cross-inhibition. Whole cell patch-clamp techniques were used to measure P2X- and nAChR-mediated currents in cultured enteric neurons and HEK-293 cells. In cultured myenteric neurons, ACh (3 mM) and ATP (1 mM) coapplication evoked an inward current that was only 57 +/- 6% (P < 0.05) of the predicted current that would have occurred if the two populations of channels were activated independently. In HEK-293 cells coexpressing alpha(3)beta(4) nAChR/P2X(2) receptors, coapplication of ATP and ACh caused a current that was 58 +/- 7% of the predicted current (P < 0.05). To test the importance of P2X subunit COOH-terminal tail length on cross-inhibition, P2X(3) and P2X(4) subunits, which have shorter COOH-terminal tails, were studied. Cross-inhibition with alpha(3)beta(4) nAChRs and P2X(3) or P2X(4) subunits was similar to that occurring with P2X(2) subunits. P2X receptor or alpha(3)beta(4) nAChR desensitization did not prevent receptor cross-inhibition. These data indicate that the alpha(3)beta(4)-P2X receptor interaction is not restricted to P2X(2) subunits. In addition, active and desensitized conformations of the P2X receptor inhibit nAChR function. These molecular interactions may modulate the function of synapses that use ATP and ACh as fast synaptic transmitters in the ENS.

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neuron's embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to alpha,beta-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to alpha,beta-methylene ATP (30 microM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of alpha,beta-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.

We studied sensitization of retrogradely labeled bladder sensory neurons and plasticity of P2X receptor function in a model of cystitis using patch-clamp techniques. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally to rats on days 0, 2, and 4. On day 5, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T12-L2) dorsal root ganglia were removed and dissociated. Bladders from CYP-treated rats showed partial loss of the urothelium and greater myeloperoxidase activity compared with controls. Bladder neurons from CYP-treated rats were increased in size (based on whole cell capacitance) compared with controls and exhibited lower activation threshold, increased action potential width, and greater number of action potentials in response to current injection or application of purinergic agonists. Most control LS bladder neurons (>85%) responded to ATP or alpha,beta-metATP with a slowly desensitizing current; these agonists affected only half of TL neurons, producing predominantly fast/mixed desensitizing currents. CYP treatment increased the fraction of TL bladder neurons sensitive to purinergic agonists (>80%) and significantly increased current density in both LS and TL bladder neurons compared with control. Importantly, LS and TL neurons from CYP-treated rats showed a selective increase in the functional expression of heteromeric P2X(2/3) and homomeric P2X(3) receptors, respectively. Although desensitizing kinetics were slower in LS neurons from CYP-treated compared with control rats, recovery kinetics were similar. The present results demonstrate that bladder inflammation sensitizes and increases P2X receptor expression and/or function for both pelvic and lumbar splanchnic pathways, which contribute, in part, to the hypersensitivity associated with cystitis.

To determine whether estrogen status modulated dorsal horn neural activity relevant to temporomandibular joint (TMJ) processing single units were recorded in superficial and deep laminae at the trigeminal subnucleus caudalis/upper cervical cord (Vc/C1-2) junction of ovariectomized (OvX) female rats under barbiturate anesthesia after 17beta-estradiol (E2) treatment for 2 days. E2 dose-dependently enhanced the response to intra-TMJ stimulation by adenosine triphosphate (ATP) of neurons classified as nociceptive specific (NS), but not wide dynamic range (WDR), in superficial laminae. ATP caused similar responses among NS and WDR neurons from deep laminae in all groups. By contrast, the cutaneous receptive field areas of WDR, but not NS, units in superficial and deep laminae were enlarged in high E2-treated (HE2) compared with low E2-treated (LE2) females. Units from untreated or vehicle-treated male rats displayed responses similar to those of LE2 females. TMJ units in superficial laminae from females were more likely to receive convergent cutaneous input and respond to jaw movement than males, independent of E2 treatment. Western blot analysis revealed similar levels of P2X2 and P2X3 receptor protein in Vc/C1-2 or trigeminal ganglion samples in all groups. Immunohistochemistry revealed dense terminal labeling for P2X3 receptors in superficial laminae and moderate labeling in deep laminae at the Vc/C1-2 junction. These data indicated a significant linkage between estrogen status and the magnitude of articular input evoked by ATP from TMJ neurons in the superficial laminae at the Vc/C1-2 junction, whereas estrogenic modulation of TMJ neurons in deep laminae affected only the convergent input from overlying facial skin.

Recent studies indicate that chemoafferent nerve fiber excitation in the rat carotid body is mediated by acetylcholine and ATP, acting at nicotinic cholinergic receptors and P2X2 purinoceptors, respectively. We previously demonstrated that, after a 10- to 14-day exposure to chronic hypoxia (CH), the nicotinic cholinergic receptor blocker mecamylamine no longer inhibits rat carotid sinus nerve (CSN) activity evoked by an acute hypoxic challenge. The present experiments examined the effects of CH (9-16 days at 380 Torr) on the expression of P2X2 purinoceptors in carotid body and chemoafferent neurons, as well as the effectiveness of P2X2 receptor blocking drugs on CSN activity evoked by hypoxia. In the normal carotid body, immunocytochemical studies demonstrated a dense plexus of P2X2-positive nerve fibers penetrating lobules of type I cells. In addition, type I cells were lightly stained, indicating P2X2 receptor expression. After CH, the intensity of P2X2 receptor immunostaining was maintained in chemosensory type I cells and in the soma of chemoafferent neurons. P2 receptor expression on type I cells was confirmed by demonstrations of ATP-evoked increased intracellular Ca2+; this response was modulated by simultaneous exposure to hypoxia. In normal preparations, CSN activity evoked by hypoxia in vitro was 65% inhibited in the presence of specific P2X2 receptor antagonists. However, unlike the absence of mecamylamine action after CH, P2X2 antagonists remained effective against hypoxia-evoked activity after CH. Our findings indicate that ATP acting at P2X2 receptors contributes to adjusted chemoreceptor activity after CH, indicating a possible role for purinergic mechanisms in the adaptation of the carotid body in a chronic low-O2 environment.

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.

P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0-P36, indicating number of days) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease (R(2) = 0.96, P

Hypocretin/orexin (hcrt) neurons play an important role in hypothalamic arousal and energy homeostasis. ATP may be released by neurons or glia or by pathological conditions. Here we studied the effect of extracellular ATP on hypocretin cells using whole cell patch-clamp recording in hypothalamic slices of transgenic mice expressing green fluorescent protein (GFP) exclusively in hcrt-producing cells. Local application of ATP induced a dose-dependent increase in spike frequency. In the presence of TTX, ATP (100 microM) depolarized the cells by 7.8 +/- 1.2 mV. In voltage clamp under blockade of synaptic activity with the GABA(A) receptor antagonist bicuculline, and ionotropic glutamate receptor antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), ATP (100 microM) evoked an 18 pA inward current. The inward current was blocked by extracellular choline substitution for Na+, had a reversal potential of -27 mV, and was not affected by nominally Ca2+-free external buffer, suggesting that ATP activated a nonselective cation current. All excitatory effects of ATP showed rapid attenuation. ATP-induced excitatory actions were mimicked by nonhydrolyzable ATP-gamma-S but not by alpha,beta-MeATP and inhibited by the purinoceptor antagonists suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS). The current was potentiated by a decrease in bath pH, suggesting P2X2 subunit involvement. Frequency and amplitude of spontaneous and miniature synaptic events were not altered by ATP. Suramin, but not PPADS, caused a small suppression of evoked excitatory synaptic potentials. Together, these results show a depolarizing response to extracellular ATP that would lead to an increased activity of the hypocretin arousal system.

Tissue inflammation contributes to the development of hyperalgesia, which is at least in part due to altered properties of primary afferent neurons. We hypothesized that gastric ulcers enhance the excitability of gastric sensory neurons and increase their response to purinergic agonists. The rat stomach was surgically exposed, and a retrograde tracer [1.1'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine methanesulfonate (DiI)] was injected into the wall of the distal stomach. Kissing ulcers (KUs) were produced by a single injection of acetic acid (0.1 ml for 45 s; 60%) into the clamped gastric lumen. Saline injection served as control. Gastric nodose ganglion (NG) or dorsal root ganglion (DRG) cells were harvested 7 days later and acutely dissociated for whole cell recordings. Based on whole cell capacitance, gastric DRG neurons exhibited larger cell size than NG neurons. Significantly more control gastric DRG neurons compared with NG counterparts had TTX-resistant action potentials. Almost all control NG neurons (90%) compared with significantly less DRG neurons (< or =38%) responded to ATP or alpha,beta-metATP. Whereas none of the control cells exhibited spontaneous activity, about 20% of the neurons from KU animals generated spontaneous action potentials. KUs enhanced excitability as shown by a decrease in threshold for action potential generation, which was in part due to an increased input resistance. This was associated with an increase in the fraction of neurons with TTX-resistant action potentials and cells responding to capsaicin and purinergic agonists. KU doubled the current density evoked by the P2X receptor agonist alpha,beta-metATP and slowed decay of the slowly desensitizing component of the current without affecting the concentration dependence of the response. These data show that KU sensitizes vagal and spinal gastric afferents by affecting both voltage- and ligand-gated channels, thereby potentially contributing to the development of dyspeptic symptoms.

The mechanism of extracellular ATP-triggered vagal depressor reflex was further studied in a closed-chest canine model. Adenosine and ATP were administered individually in equimolar doses (0.01-1.0 mumol/kg) into the right coronary artery (RCA) and left circumflex coronary artery (LCA). When administered into the RCA, adenosine and ATP exerted an identical and relatively small negative chronotropic effect on sinus node automaticity; the time to peak negative chronotropic effect was >/=7 s. When administered into the LCA, adenosine had no effect on sinus node automaticity, whereas ATP markedly suppressed sinus node automaticity. This effect of ATP 1) reached its peak in <2 s after its administration, 2) was short lasting, and 3) was completely abolished by either intravenous administration of the muscarinic cholinergic blocker atropine (0.2 mg/kg) or intra-LCA administration of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a potent P2X(2/3) purinergic receptor (P2X(2/3)R) antagonist, but not by diinosine pentaphosphate (Ip(5)I), a potent inhibitor of P2X(1)R and P2X(3)R. Repetitive administrations of ATP were not associated with reduced effects, indicative of receptor desensitization, thereby excluding the involvement of the rapidly desensitized P2X(1)R in the action of ATP. It was concluded that ATP triggers a cardio-cardiac vagal depressor reflex by activating P2X(2/3)R located on vagal sensory nerve terminals localized in the left ventricle. Because these terminals mediate vasovagal syncope, these data could suggest a mechanistic role of extracellular ATP in this syndrome and, in addition, give further support to the hypothesis that endogenous ATP released from ischemic myocytes is a mediator of atropine-sensitive bradyarrhythmias associated with left ventricular myocardial infarction.

ATP contributes to mechanosensory transduction in the rat colorectum. P2X3 receptors are present on dorsal root ganglia (DRG) neurons that supply this area of the gut. Previous studies have shown an increased role for ATP in inflamed tissues. We aimed to investigate whether an increased purinergic component exists during mechanosensory transduction in a rat model of colitis. An in vitro rat colorectal preparation was used to investigate whether distension increased ATP release and to evaluate the role of purinergic antagonists in distension-evoked sensory discharges in the pelvic nerve in normal and colitis preparations. DRG neuron purinoceptors were also studied. Distension-evoked responses in the colitis model were attenuated to a significantly greater extent by 2',3'-O-trinitrophenyl-ATP and pyridoxyl 5-phosphate 6-azophenyl-2',4'-disulfonic acid. Inflammation caused augmented distension-evoked sensory nerve excitation after application of ATP and alpha,beta-methylene ATP. Single-fiber analysis confirmed that mean firing per unit was increased. Distension-evoked increases in ATP release from epithelial cells were substantially higher. The number of DRG neurons responding to ATP and the number of those staining for the P2X3 receptor, particularly those containing calcitonin gene-related peptide, were increased. Adenosine, after ectoenzymatic breakdown of ATP, is involved to a lesser degree in the longer-lasting distension-evoked sensory discharge, suggesting reduced ATPase activity. It was therefore concluded that ATP has an enhanced role in mechanosensory transduction in the inflamed rat colorectum. The underlying mechanisms appear to involve increased distension-evoked release of ATP as well as an increase in the number of DRG neurons supplying the colorectum expressing P2X3 receptors, especially those containing calcitonin gene-related peptide.

On the basis of the high level of P2X receptor expression found in phrenic motoneurons (MN) in rats (Kanjhan et al., J Comp Neurol 407: 11-32, 1999) and potentiation of hypoglossal MN inspiratory activity by ATP (Funk et al., J Neurosci 17: 6325-6337, 1997), we tested the hypothesis that ATP receptor activation also modulates phrenic MN activity. This question was examined in rhythmically active brain stem-spinal cord preparations from neonatal rats by monitoring effects of ATP on the activity of spinal C4 nerve roots and phrenic MNs. ATP produced a rapid-onset, dose-dependent, suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium-sensitive increase in C4 root tonic discharge and a 22 +/- 7% potentiation of inspiratory burst amplitude. This was followed by a slower, 10 +/- 5% reduction in burst amplitude. ATPgammaS, the hydrolysis-resistant analog, evoked only the excitatory response. ATP induced inward currents (57 +/- 39 pA) and increased repetitive firing of phrenic MNs. These data, combined with persistence of ATP currents in TTX and immunolabeling for P2X2 receptors in Fluoro-Gold-labeled C4 MNs, implicate postsynaptic P2 receptors in the excitation. Inspiratory synaptic currents, however, were inhibited by ATP. This inhibition differed from that seen in root recordings; it did not follow an excitation, had a faster onset, and was induced by ATPgammaS. Thus ATP inhibited activity through at least two mechanisms: 1) a rapid P2 receptor-mediated inhibition and 2) a delayed P1 receptor-mediated inhibition associated with hydrolysis of ATP to adenosine. The complex effects of ATP on phrenic MNs highlight the importance of ATP as a modulator of central motor outflows.

Extracellular ATP elevates cytosolic Ca(2+) by activating P2X and P2Y purinoceptors and voltage-sensitive Ca(2+) channels (VCCCs) in PC-12 cells, thereby facilitating catecholamine secretion. We investigated the mechanism by which ATP activates VSCCs. 2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP) and UTP were used as preferential activators of P2X and P2Y, respectively. Nifedipine inhibited the ATP- and 2-MeS-ATP-evoked cytosolic Ca(2+) concentration increase and [(3)H]norepinephrine secretion, but not the UTP-evoked responses. Studies with Ca(2+) channel blockers indicated that L-type VSCCs were activated after the P2X activation. Mn(2+) entry profiles and studies with thapsigargin revealed that Ca(2+) entry, rather than Ca(2+) release, was sensitive to nifedipine. Although P2X(2) and P2X(4) receptor mRNAs were detected, studies with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid revealed that P2X(2) was mainly coupled to the L-type VSCCs. The inhibitory effect of nifedipine did not occur in the absence of extracellular Na(+), suggesting that Na(+) influx, which induces depolarization, was essential for the P2X(2)-mediated activation of VSCCs. We report that depolarization induced by Na(+) entry through the P2X(2) purinoceptors effectively activates L-type VSCCs in PC-12 cells.

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.

ATP has been suggested to act as a neurotransmitter or a neuromodulator in the cochlea. The responses to ATP in different cell types of the cochlea vary in terms of the rate of desensitization and magnitude, suggesting that there may be different subtypes of P2X receptors distributed in the cochlea. Recently three ionotropic P2X(2) receptor splice variants, P2X(2-1), P2X(2-2), and P2X(2-3,) were isolated and sequenced from a guinea pig cochlear cDNA library. To test the hypothesis that these different splice variants could be expressed as functional homomeric receptors, the three P2X(2) receptor variants were individually and transiently expressed in human embryonic kidney cells (HEK293). The biophysical and pharmacological properties of these receptors were characterized using the whole cell patch-clamp technique. Extracellular application of ATP induced an inward current in HEK293 cells containing each of the three splice variants in a dose-dependent manner indicating the expression of homomeric receptors. Current-voltage (I-V) relationships for the ATP-gated current show that the three subtypes of the P2X(2) receptor had a similar reversal potential and an inward rectification index (I(50 mV)/I(-50 mV)). However, the ATP-induced currents in cells expressing P2X(2-1) and P2X(2-2) variants were large and desensitized rapidly whereas the current in those cells expressing the P2X(2-3) variant was much smaller and desensitized slower. The order of potency to ATP agonists was 2-MeSATP > ATP > alpha,beta -MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on all three variants. Results demonstrate that three P2X(2) splice variants from guinea pig cochlea, P2X(2-1), P2X(2-2), and P2X(2-3), can individually form nonselective cation receptor channels when these subunits are expressed in HEK293 cells. The distinct properties of these P2X(2) receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.

Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.

The effects of ATP and other nucleotides on the cytosolic Ca2+ concentration ([Ca2+]i) of single immunocytochemically typed anterior pituitary (AP) cells have been studied. ATP increased [Ca2+]i in a large percentage (60-88%) of all five AP cell types: lactotropes, somatotropes, corticotropes, gonadotropes, and thyrotropes. Additivity experiments suggest the presence of at least two different receptors, one accepting both ATP and UTP (U receptor), producing Ca2+ release from the intracellular stores, and the other preferring ATP (A receptor), producing Ca2+ (and Mn2+) entry. The characteristics of the U and A receptors were consistent with those of P2Y2 and P2X2, respectively, and their distribution in the different AP cell types was not homogeneous. The presence of other ATP receptors such P2Y1 or P2X2/P2X3 heteropolymers in a small fraction of the cells cannot be excluded. Thus functional ionophoric P2X receptors, which are typical of neural tissue, are also present in the pituitary gland and could contribute to regulation of the gland's function.