Enhancement of ATP-activated current by protons in dorsal root ganglion neurons

 The effect of pH on ATP-activated current in bullfrog dorsal root ganglion neurons was studied using the whole-cell patch-clamp technique. ATP-activated current amplitude was highly dependent upon extracellular pH. An acid pH increased, whereas alkaline pH decreased, ATP-activated current amplitude. The half-maximal pH (EC₅₀) for potentiation of 2.5 μM ATP-activated current was 7.2. Acidification alone did not activate detectable current and, at an acid pH, ATP-activated current was abolished by suramin. Proton-induced enhancement of ATP-activated current was not sensitive to membrane potential between –80 and +40 mV, and did not involve a shift in reversal potential. Lowering pH from 7.2 to 6.5 or elevating pH from 7.2 to 8.0 shifted the ATP concentration/response curve to the left or right, respectively, without changing the maximal response to ATP. Protons increased the time constant of deactivation without affecting the time constant of activation or desensitization of ATP-activated current. Alteration of patch-pipette (intracellular) pH did not affect the enhancement of ATP-activated current by extracellular protons. Diethylpyrocarbonate (DEP), dithiothreitol (DTT), 5,5′-dithio-bis-(2-nitro-benzoic acid) (DTNB), or N-ethylmaleimide (NEM) did not affect enhancement of ATP-activated current by protons. The results suggest that extracellular protons, at physiological concentrations, can regulate the function of P_2X purinoceptors by modulating the affinity of the ATP-binding site. Received: 20 July 1996 / Received after revision and accepted: 24 September 1996     

Direct excitation of hypocretin/orexin cells by extracellular ATP at P2X receptors

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.     

Zn2+ potentiates ATP-activated currents in rat sympathetic neurons

The ATP-activated inward current (I _ATP) in cultured rat superior cervical ganglion neurons and its modulation by extracellular Zn²⁺ were examined. ATP activated a non-specific cation conductance and caused a transient rise in intracellular Ca²⁺. The current response was specifically activated by ATP and was blocked by the P₂-purinoceptor antagonist, suramin. Low concentrations of extracellular Zn²⁺ rapidly and reversibly potentiated both I _ATP and the intracellular Ca²⁺ rise. The potentiation by 10 M Zn²⁺ was dependent on the concentration of agonist; Zn²⁺ increased the sensitivity of activation without potentiating the maximum response. Higher concentrations of Zn²⁺ reduced and prolonged the current, consistent with open-channel block. We hypothesize that there exist two sites of action for Zn²⁺: a positively acting allosteric site that enhances current amplitude and a site, possibly within the pore, that blocks conductance through the channel.     

Adenosine triphosphate-activated inward current in isolated smooth muscle cells from rat vas deferens

The electrophysiological effect of adenosine triphosphate (ATP) on the enzymatically dispersed smooth muscle cells from rat vas deferens was investigated. ATP always induced depolarization accompanied with a reduction in membrane resistance. In a whole cell voltage clamp experiment, an inward current was recorded when the cell was exposed to ATP-containing solution. The ATP-induced current disappeared within 2min even in the continuous presence of ATP, which may indicate that the cells were desensitized to this compound. The ATP-induced current was also recorded in the cells superfused with 10^–5M nicardipine or in the Cs-loaded cells, eliminating the possible involvement of voltage-gated Ca and K current. During cell-attached patch clamp, an elementary current having a mean conductance of 20pS was observed when the intrapipette solution was changed to ATP-containing solution. The estimated zero current potentials of the ATP-activated macroscopic current and elementary current were about 0mV. These results suggest that ATP exerts its transmitter-like action by activating ion channels in smooth muscles.     

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels

In airway smooth muscle (ASM), adenosine 5′-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K⁺ currents (I _K) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I _K increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I _K was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I _K increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y₂ and P2Y₄ receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y₄ and P2Y₂ receptors; (2) induces I _K increment through activation of the delayed rectifier K⁺ channels and the high-conductance Ca²⁺-dependent K⁺ channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I _K increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.     

Roles of Ca2+ influx through ATP-activated channels in catecholamine release from pheochromocytoma PC12 cells.

1. Extracellular ATP evokes catecholamine release concomitant with depolarization in pheochromocytoma PC12 cells. Roles of Ca2+ influx through ATP-activated channels during the catecholamine release were investigated. 2. Norepinephrine or dopamine release induced by > or = 100-microM concentrations of ATP was insensitive to 300 microM Cd2+, whereas the release induced by increasing extracellular KCl (50-150 mM) was completely blocked by this concentration of Cd2+. 3. ATP (100 microM) increased the intracellular free Ca2+ concentration measured with fura-2. The increase was not affected by 300 microM Cd2+ or 100 microM nicardipine, suggesting that Ca2+ influx through ATP-activated channels but not through voltage-gated Ca2+ channels contributes to the ATP-evoked catecholamine release. 4. Inward currents permeating through voltage-gated Ca2+ channels were measured using the whole-cell voltage clamp. In the presence of 10 microM ATP, a concentration that induces an ATP-activated channel-mediated current equivalent to that induced by 100 microM ATP during the depolarization in "non-voltage clamped" cells, the Ca2+ current activated by a voltage step to +10 mV was reduced. The reduction in the Ca2+ channel-mediated current was not observed when the extracellular Ca2+ was replaced with Ba2+. 5. The ATP (100 microM)-evoked dopamine release was inhibited by 300 microM Cd2+ when measured with extracellular Ba2+ instead of Ca2+. This effect of Ba2+ may not be related to K+ channel-blocking activity, because the ATP-evoked dopamine release obtained with 5 mM tetraethylammonium (TEA) was not inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠三叉神经节神经元ATP-激活电流的药理学特性

本实验应用全细胞膜片钳技术探讨了大鼠三叉神经节神经元ATP-激活电流的药理学特性。结果显示:(1)92.3%(60/65)的细胞对ATP敏感,有反应的细胞可记录到三种型式的ATP-激活电流:快速激活快速失活型(Fasttype,F型)、快速激活缓慢失活型(Intermediate type,I型)和缓慢激活缓慢失活型(Slow type,S型),三种电流均具有浓度依赖性。(2)动力学特征:三种类型的ATP激活电流上升相从10%到90%的时间:F型:33.6±4.5ms;I型:62.2±9.9ms;S型:302.1±62ms。去敏感相从10%到90%的时间:F型:399.4±58.2ms;S型:>500ms。(3)量-效关系:I型的量-效曲线居中间,F型的下移,S型的上移,三种类型电流的量-效曲线的EC50非常接近。(4)ATP受体的激动剂激活三种类型电流的效能顺序为:ATP>2-MeSATP>α,β-MeATP>UTP>ADP,其拮抗剂PPADS、RB-2和Suramin对这三种电流的拮抗作用基本相同。以上结果提示:三种型式的ATP-激活电流可能是由不同亚单位组合的P2X受体亚型所介导,不同的亚型介导的电流可能具有不同的功能。     

Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells

Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (K _d = 15.4 ± 4.6 μM; B _max = 1.7 ± 0.5 nmol mg⁻¹ protein); and a low affinity site (K _d = 170 ± 94.5 μM; B _max = 14.2 ± 3.9 nmol mg⁻¹ protein). In myotube membranes only a low affinity binding site for GTP (K _d = 169 ± 39 μM; B _max = 12.3 ± 1.4 nmol mg⁻¹ protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca²⁺]_i. The patterns of Ca²⁺ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca²⁺ responses: (1) a fast increase in [Ca²⁺]_i, followed by a sustained [Ca²⁺]_i elevation, and (2) a slow raising and steady prolonged increase in [Ca²⁺]_i. In myotubes, however only fast Ca²⁺ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca²⁺]_i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca²⁺ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dual control by ATP and acetylcholine of inwardly rectifying K+ channels in bovine atrial cells

Whole-cell and single-channel recordings were used to study an ionic current activated by extracellular adenosine 5′-triphosphate (ATP) applied to calf atrial cells. ATP (K _d ≈ 10μM) elicited an inwardly rectifying current that reversed nearE _K and was blocked by external Cs⁺ (10 mM). Under identical conditions, adenosine had no effect. Cell-attached patch recordings revealed an ATP-activated channel with a slope conductance of about 30 pS. At both the whole-cell and single-channel levels, the channels activated by ATP seemed nearly identical to the potassium channels activated by acetylcholine (ACh) in the same cells. However, the effects of ATP were not affected by atropine, suggesting that ATP does not interact with the same receptors as ACh. In some cells, whole-cell currents of similar magnitude were activated by ACh alone, ATP alone, or ACh and ATP applied together. These results suggest that calf atrial cells possess a population of inwardly rectifying potassium channels that are controlled jointly by two populations of receptors selective for ACh and ATP.     

In airways ATP refills sarcoplasmic reticulum via P2X smooth muscle receptors and induces contraction through P2Y epithelial receptors

In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca(2+)](i). Cytosolic Ca(2+) is extruded to the extracellular space by the Na(+)/Ca(2+) exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCX(REV)), inducing Ca(2+) entry. We investigated whether ATP, via P2X receptors, activates the NCX(REV) and whether the increment in [Ca(2+)](i) is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca(2+)](i) not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca(2+)](i), (a Ca(2+) peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca(2+) peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca(2+)](i) increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCX(REV) and this effect was abolished by SKF 96365 and TNP-ATP (P2X(1) and P2X(3) receptors antagonist). P2X(1) and P2X(3) receptors were corroborated by immunoblotting of ASM. NCX(REV) activation and ATP in the presence of RB2 favor the SR Ca(2+) refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca(2+)](i) through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X(1) and P2X(3) receptors and the NCX(REV) which refills the SR.     

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells

We have studied the pathways by which extra-cellular bradykinin and adenosine 5-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca²⁺]_i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca²⁺]_i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca²⁺ mobilization from an internal pool followed by a sustained increase in [Ca²⁺]_i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca²⁺ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca²⁺]_i, due to an increase in the driving force for Ca²⁺ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca²⁺]_i. Inward current and [Ca²⁺]_i were enhanced during hyperpolarizing steps indicating a substantial Ca²⁺ influx through ATP-activated channels. No release of Ca²⁺ from internal stores, but a large Na⁺ inward current, was observed in Ca²⁺-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.     

Suramin inhibits the mixed lymphocyte reaction by suppressing lymphokine production

New compounds with a greater potency than cyclosporin A (CyA) for thwarting host rejection of organ transplantation are being sought. Suramin sodium may be a novel drug to prevent or delay graft rejection and graft-vs-host disease (GVHD), because of itsin vitro andin vivo immunosuppressive properties. Since the allogeneic mixed lymphocyte reaction (MLR) is considered to be thein vitro counterpart of the initial T-lymphocyte recognition and response to allogeneic histocompatibility antigens on grafted tissue or organ and to GVHD, we initially evaluated thein vitro suppressive effect of suramin in the allogeneic MLR. Suramin inhibited the H-2- and HLA-incompatible MLR in a dose-dependent manner. The suppressive effect was observed both in the primary and in the secondary MLR. The suppression of the MLR by suramin is due predominantly to the inhibition of interleukin-2 (IL-2) production by the responding T cells.     

不同初级感觉神经元ATP-激活电流的特征及意义

为探讨大鼠不同初级感觉神经元ATP-激活电流的电生理学特征及意义,本实验在新鲜分离的大鼠三种初级感觉神经元标本上,应用全细胞膜片钳技术记录,对外加ATP引起的膜电流的变化进行了研究。结果显示:(1)受检的细胞绝大多数对ATP敏感,外加ATP记录到内向电流,在背根神经节和三叉神经节神经元记录到三种形式的ATP-激活电流:快速激活快速失活型(fasttype,F型)、快速激活缓慢失活型(intermediatetype,I型)和缓慢激活缓慢失活型(slowtype,S型),但在结状神经节神经元主要记录到S型电流,三种形式的电流均具有浓度依赖性;(2)三种形式的ATP-激活电流上升相(risingphase,R)从10%至90%的时间(R10-90)有明显差异(P<0.05),去敏感相(desensitizingphase)从10%至90%的时间(D10-90)也有明显差异(P<0.05);(3)三种形式的电流EC50非常接近,I型的量-效曲线居中间,F型的下移,S型的上移;(4)小细胞的ATP-激活电流多表现为F型特征,大细胞多表现为S型特征,而中等大小的细胞多表现为I型特征,结状神经节神经元电流形式与细胞大小没有相关性。以上结果提示:躯体和内脏初级感觉神经元ATP-激活电流的类型可能与不同的感觉性质有关;不同形式的ATP激活电流可能由ATP受体的不同亚单位组成的各种亚型中介。     

Novel mechanism of inhibition by the P2 receptor antagonist PPADS of ATP-activated current in dorsal root ganglion neurons

The antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) has been proposed to selectively antagonize the actions of ATP at P2X receptors. Whole cell patch-clamp recording techniques therefore were used to characterize PPADS inhibition of ATP-activated current in bullfrog dorsal root ganglion (DRG) neurons. PPADS, 0.5-10 microM, inhibited ATP-activated current in a concentration-dependent manner with an IC(50) of 2.5 +/- 0.03 microM. PPADS produced a gradual decline of ATP-activated current to a steady state, but this was not an indication of use dependence as the gradual declining component could be eliminated by exposure to PPADS before ATP application. In addition, ATP-activated current recovered completely from inhibition by PPADS in the absence of agonist. The slow onset of inhibition by PPADS was not apparently due to an action at an intracellular site as inclusion of 10 microM PPADS in the recording pipette neither affected the ATP response nor did it alter inhibition of the ATP response when 2.5 microM PPADS was applied externally. PPADS, 2.5 microM, decreased the maximal response to ATP by 51% without changing its EC(50). PPADS inhibition of ATP-activated current was independent of membrane potential between -80 and +40 mV and did not involve a shift in the reversal potential of the current. The magnitude of PPADS inhibition of ATP-activated current was dependent on the duration of the prior exposure to PPADS. The time constants of both onset and offset of PPADS inhibition of ATP-activated current did not differ significantly with changes in ATP concentration from 1 to 5 microM. Recovery of ATP-activated current from PPADS inhibition also exhibited a slow phase that was not accelerated by the presence of agonist and was dependent on the concentration of PPADS. The apparent dissociation rate of PPADS from unliganded ATP-gated ion channels was much greater than the rate of the slow phase of recovery of ATP-activated current from PPADS inhibition. The results suggest that PPADS can inhibit P2X receptor function in a complex noncompetitive manner. PPADS produces a long-lasting inhibition that does not appear to result from open channel block but rather from an action at an allosteric site apparently accessible from the extracellular environment that involves a greatly reduced rate of dissociation from liganded versus unliganded ATP-gated ion channels.     

ATP released from astrocytes during swelling activates chloride channels

ATP release from astrocytes contributes to calcium ([Ca(2+)]) wave propagation and may modulate neuronal excitability. In epithelial cells and hepatocytes, cell swelling causes ATP release, which leads to the activation of a volume-sensitive Cl(-) current (I(Cl,swell)) through an autocrine pathway involving purinergic receptors. Astrocyte swelling is counterbalanced by a regulatory volume decrease, involving efflux of metabolites and activation of I(Cl,swell) and K(+) currents. We used whole cell patch-clamp recordings in cultured astrocytes to investigate the autocrine role of ATP in the activation of I(Cl,swell) by hypo-osmotic solution (HOS). Apyrase, an ATP/ADP nucleotidase, inhibited HOS-activated I(Cl,swell), whereas ATP and the P2Y agonists, ADPbetaS and ADP, induced Cl(-) currents similar to I(Cl,swell). Neither the P2U agonist, UTP nor the P2X agonist, alpha,beta-methylene ATP, were effective. BzATP was less effective than ATP, suggesting that P2X7 receptors were not involved. P2 purinergic antagonists, suramin, RB2, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) reversibly inhibited activation of I(Cl,swell), suggesting that ATP-activated P2Y1 receptors. Thus ATP release mediates I(Cl,swell) in astrocytes through the activation of P2Y1-like receptors. The multidrug resistance protein (MRP) transport inhibitors probenicid, indomethacin, and MK-571 all potently inhibited I(Cl.swell). ATP release from astrocytes in HOS was observed directly using luciferin-luciferase and MK-571 reversibly depressed this HOS-induced ATP efflux. We conclude that ATP release via MRP and subsequent autocrine activation of purinergic receptors contributes to the activation of I(Cl,swell) in astrocytes by HOS-induced swelling.     

Luminal ATP-induced contraction of rabbit pulmonary arteries and role of purinoceptors in the regulation of pulmonary arterial pressure

The effects of luminal ATP between rabbit pulmonary (PAs) and coronary arteries (CAs) were compared to understand the role of purinoceptors in the regulation of pulmonary arterial pressure (PAP) under hypoxia. Diameters of vessels were video analyzed under luminal perfusion. ATP-induced membrane currents and intracellular Ca²⁺ signals ([Ca²⁺]_i) were compared in pulmonary (PASMCs) and coronary myocytes (CASMCs) using patch clamp and spectrofluorimetry. PAP was measured in perfused lungs under ventilation. Luminal ATP induced constriction of rabbit PAs in the presence of endothelium. In contrast, CAs showed dilating responses to luminal ATP even in the absence of endothelium. In PASMCs, both P2X-mediated inward current and P2Y-mediated store Ca²⁺ release were consistently observed. In contrast, CASMCs showed neither P2X nor P2Y responses. In the perfused lungs, hypoxia-induced PAP increase was decreased by suramin, a purinergic antagonist. A luminal application of α,β-meATP largely increased PAP, whereas UTP decreased PAP. The combined application of P2X- and P2Y-selective agonists (α,β-meATP and UTP) increased PAP. However, the perfusion of ATP alone decreased PAP, and the ATP-induced PAP decrease was affected neither by adenosine receptor antagonist nor by nitric oxide synthase inhibitor. In summary, although the luminal ATP constricts isolated PAs and suramin attenuated the HPV of perfused lungs, the bimodal responses of PAP to purinergic agonists indicate that the luminal ATP regulates pulmonary circulation via complex signaling interactions in situ.     

Differential modulation by copper and zinc of P2X2 and P2X4 receptor function.

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.     

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A₂ (TXA₂) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA₂ production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA₂ production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA₂ dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.     

Effect of suramin on phosphorylation-dephosphorylation reactions in Trypanosoma gambiense

Suramin which is used as a chemotherapeutic agent against sleeping sickness was tested for its ability to inhibit phosphorylation-dephosphorylation reactions in Trypanosoma gambiense. Protein-kinase I, which is characterized by its preference for acidic acceptor proteins, was found to be strongly inhibited by suramin. The inhibition constant was calculated to be 0.8 μM. The type of inhibition was non-competitive with respect to ATP as well as to substrate protein.