P2X₇ Receptor Activation–Induced Contraction and Lysis in Human Saphenous Vein Smooth Muscle

From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UPR 411, Valbonne, France.

Correspondence to Pierre Pacaud, PhD, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UPR 411, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail pacaud{at}ipmc.cnrs.fr

	Abstract

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Abstract—In cutaneous veins where purinergic neurotransmission is more prominent compared with in deep vessels, physiological and pathological roles of nerve-released ATP have been described. Neuronally released ATP has been reported to act through activation of unidentified ionotropic P2X receptor(s). This study analyzed P2X receptor subtypes expressed in human saphenous vein smooth muscle and their physiological functions. Transcripts for both hP2X₁ receptors, already identified in other smooth muscles, and, surprisingly, hP2X₇ receptors known to be responsible for the cytotoxic effect of ATP in macrophages were detected by Northern blot analysis in total RNA from saphenous vein smooth muscle. ATP and other P2X receptor agonists [ß-methylene-ATP, 2-methylthio-ATP, and 2',3'-(4-benzoyl)benzoyl-ATP] dose-dependently contracted venous rings, but the contraction induced by 2-methylthio-ATP was more transient than that evoked by the other P2X agonists. The effect of hP2X₁ agonists involved the activation of a rapidly desensitizing cation current recorded in freshly isolated myocytes. The action of hP2X₇ receptor agonists was related to a maintained nondesensitizing cation current. In addition, hP2X₇ receptor activation formed membrane pores that were permeable to large molecules. hP2X₁ and hP2X₇ receptors coexpressed in COS cells did not associate to form heteromultimers. Our data indicate that both hP2X₁ and hP2X₇ receptors are expressed as 2 separated populations of channels in human saphenous vein myocytes and are involved in ATP-induced tension. We suggest that cell lysis consequent to hP2X₇ receptor–induced pore formation contributes to the disorganization and decrease in the amount of contractile myocytes in the media of varicose veins.

Key Words: cation channel • purinoceptor • vein • smooth muscle

	Introduction

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In cutaneous vessels where purinergic neurotransmission is more prominent compared with in deep vessels, physiological or pathophysiological roles of nerve-released ATP have been suggested.¹ ATP released from presynaptic nerves causes typical fast excitatory postsynaptic potential in smooth muscle.^{2 3} Neuronally released ATP is involved in the contractile response of cutaneous vein on exposure to cold⁴ and has been shown to exert a trophic action on venous myocytes.⁵

The postjunctional action of ATP released from nerve terminals is ascribed to activation of P2X receptors. P2X receptors constitute a family of ligand-gated ion channels that bind extracellular ATP, structurally distinct from other ligand-gated channels. These receptors are proteins that have intracellular amino and carboxy termini and a large extracellular loop between 2 hydrophobic segments.⁶ Seven different P2X receptors have now been cloned. The P2X₁ receptor cDNA was originally isolated from smooth muscle.⁷ The 6 other members of the family were essentially cloned from nervous tissue.⁸ These 7 receptors can be further divided in 2 groups by comparison of functional properties.⁹ P2X₁ and P2X₃ form the first group, characterized by a rapid desensitization in the continuous presence of ATP and a high sensitivity to ß-MeATP. P2X₂ and P2X₄ through P2X₇ do not desensitize and are not sensitive to ß-MeATP. P2X₇ is considered to represent the previously termed P_2Z receptor responsible for ATP-dependent lysis of macrophages.¹⁰ P2X₇ displays particular properties: (1) its activation requires high ATP concentrations and BzATP is the most effective agonist, (2) P2X₇-induced responses are strongly potentiated by removal of extracellular Mg²⁺ and/or Ca²⁺, and (3) P2X₇ receptor activation can form membrane pores permeable to large molecules.¹¹

In native tissues, the functional properties of P2X receptors do not always correspond to those found for receptors expressed in heterologous cells, suggesting that the native P2X channels might form either homomultimers or heteromultimers of cloned subunits or that they contain additional subunits not yet cloned.¹²

Depending on the P2X subunits expressed in venous smooth muscle, ATP could thus have several different actions. Cloning of human P2X (hP2X) subunits now provides the possibility to precisely analyze the effects of ATP and to consider its physiological and/or pathological roles in the human venous wall. In freshly isolated smooth muscle cells from human saphenous vein, we have previously shown that ATP elicited a nonselective cation current and induced a rise in intracellular Ca²⁺ concentration through activation of an unidentified P2X receptor.¹³ The aim of the present study was to identify by a molecular approach the P2X receptor subtypes expressed in smooth muscle of human saphenous veins and to analyze their physiological functions.

	Materials and Methods

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Tissue Preparation
Human saphenous veins were obtained from 35 patients (27 women and 8 men; mean age, 52.7±1.9 years) undergoing surgical removal of varicose veins. In the operating room, veins were immediately placed in ice-cold PSS (see "Solutions" for the composition) and rapidly transported to the laboratory. All the procedures followed were in accordance with institutional guidelines.

Damaged or dilated parts of the veins were discarded. The remaining parts of the veins were cleaned of adherent connective tissue. The endothelium was carefully removed by gently rubbing the intimal surface with the tip of small forceps. The vessels were then prepared for tension measurement, RNA extraction, or isolated cell preparation.

Contraction Measurement in Intact Smooth Muscle
The cleaned veins were cut into rings (5 to 7 mm in length, 1 mm wide) that were suspended under isometric conditions and connected to a force transducer (Gould) in 3-mL organ baths filled with Krebs-Henseleit solution, maintained at 31°C, and gassed with 95% O₂/5% CO₂. The preparations were initially placed under a resting tension of 1 g, left to equilibrate for 1 hour, and washed at 20-minute intervals. Successive applications of agonist were separated by a time interval of 20 minutes. Concentration-response curves to agonists were obtained by increasing the concentration in the organ chamber. All contractile responses were expressed as percentages of the maximal response elicited by 10 µmol/L noradrenaline.

RT-PCR Analysis
Total RNA was extracted from human saphenous vein by use of Chirgwin's method. Reverse transcription was carried out using 1 µg total RNA, 500 ng oligo(dT)₁₅ (Promega), 0.2 mmol/L dNTPs, and 200 U per assay of Superscript MMLV (GIBCO BRL) according to the protocol of the manufacturer.

PCR was carried out using 1/10 of the reverse transcription. Taq DNA polymerase was from Promega (1 U per 25 µL assay). Specific primers designed to amplify the hP2X₁ receptor wereas follows: sense, 5'-CCCACCATGGCACGGCGGT-3'; antisense, 5'-CCAACCACTCCACCCTTCTCA-3'; the product was a 767-bp fragment. The hP2X₇ receptor was amplified using the following primers: sense, 5'-CTGCTGTCGCTCCCATATT-3'; antisense, 5'-CTGTACTGCCCTTCACTCT-3'; the product was a 684-bp fragment. Primers used to amplify the hP2X₃ receptor were those reported by Garcia-Guzman et al.¹⁴ The fragments were subcloned into the SmaI site of a BlueScript SK vector and sequenced using the dideoxy terminator sequencing method.

Northern Blot Analysis
Total RNA (15 µg per lane) was fractionated onto a 1.3% agarose, 2.2 mol/L formaldehyde gel and transferred to Nytran N+ membranes (Schleicher & Schuell) before hybridization with the selected probes. The hP2X₁ and hP2X₇ probes were the PCR products described above. The GAPDH probe was a 1.1-kb cDNA corresponding to the rat sequence and was kindly provided by F. Moreau-Gachelin (Paris, France). The probes were labeled with [-³²P]dCTP (ICN) using the Prime-a-Gene labeling system (Promega). Hybridization was carried out overnight at 45°C in a buffer containing 50% formamide, 5x SSPE, 5x Denhardt's solution, 0.1% SDS, and 100 µg/mL denatured salmon sperm DNA. Membranes were then washed twice with 2x SSPE, once with 1x SSPE, and once with 0.5x SSPE for 30 minutes at 55°C. Hybridization signals were analyzed with a phosphorimager (Fugi). Dehybridization of the membrane was performed before the blot was probed with a different probe, and the absence of signal was checked.

Expression of P2X Receptors in COS Cells
Human urinary bladder hP2X₁ cDNA (GenBank accession number X83688) subcloned into the pBKCMV expression vector and human monocyte hP2X₇ cDNA (GenBank accession number Y09561) subcloned into pcDNA3 expression vector were kindly supplied by Dr Gary Buell (Glaxo Institute for Molecular Biology, Geneva, Switzerland). COS cells were seeded at a density of 20 000 cells per 35-mm diameter dish 24 hours before transfection. Cells were then transfected with 1 µg cDNA (hP2X₁, hP2X₇) and 0.5 µg of CD8 plasmids using 0.5 mg/mL diethylaminoethyl-dextran in 200 µL PBS. After incubation for 30 minutes at 37°C, 2 mL DMEM (GIBCO BRL) containing 10% FCS and 80 µmol/L chloroquine were added. After a 3-hour incubation at 37°C, the medium was discarded and replaced by DMEM containing 10% FCS and 10% DMSO and left for 2 minutes. Cells were then washed, placed in fresh DMEM containing 10% FCS, and maintained in an incubator gassed with 95% air/5% CO₂ at 37°C. Cells were used for electrophysiological or fluorescence measurements 48 to 72 hours later. Transfected cells were visualized using the anti-CD8 antibody–coated bead method.¹⁵ In control experiments, we verified that ATP and BzATP did not induce change in the membrane current of untransfected COS cells.

Smooth Muscle Cell Preparation
The saphenous vein was dissected from adventitia and opened longitudinally; it was then cut into small pieces, washed for 10 minutes in low Ca²⁺ (40 µmol/L) PSS, and incubated in low-Ca²⁺ PSS containing 1 mg/mL collagenase, 0.5 mg/mL pronase, 0.06 mg/mL elastase, and 1 mg/mL BSA at 37°C for 30 minutes. After this time, the solution was removed, and the pieces of veins were incubated again in a fresh enzyme solution at 37°C for 30 minutes. The pieces were then placed in enzyme-free solution and triturated using a fire-polished Pasteur pipette to separate the cells, which were stored on glass coverslips at 4°C in PSS containing 0.8 mmol/L Ca²⁺ and used on the same day.

Membrane Current Measurement
Whole-cell membrane current recordings were made at room temperature with standard patch-clamp techniques using a Biologic RK400 patch-clamp amplifier (Biologic Co) with borosilicate patch pipettes of 1 to 4 M resistance. The series resistances (5 to 8 M) were not corrected. The liquid junction potentials were corrected with an offset circuit before each experiment. Axon DigiData 1200 interface and pClamp 6 software (Axon Instruments) were used to generate voltage-clamp protocols and for data acquisition (Clampex). Agonists were applied by high-speed perfusion of the experimental chamber or by pressure ejection from glass pipettes placed near the recorded cell.

Measurement of Ethidium Fluorescence
The ability of BzATP to cause permeabilization of cells, with subsequent uptake of ethidium bromide, was tested by incubating coverslips with attached cells in Mg²⁺-free solution containing 25 µmol/L ethidium bromide. Fluorescence intensity of cells was monitored using a Nikon Diaphot inverted microscope fitted with an epifluorescence attachment (Nikon France). The cell studied was illuminated at 360 nm. Emitted light was counted at 580 nm with a photomultiplier (P1, Nikon). The voltage signals from the photomultiplier were stored in an IBM-PC computer for subsequent analysis.

Solutions
The PSS contained (in mmol/L) 130 NaCl, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 11 glucose, and 10 HEPES, brought to pH 7.4 with NaOH. For tension measurements, the Krebs-Henseleit solution had the following composition (in mmol/L): 118.4 NaCl, 4.7 KCl, 2 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, and 11 glucose. For the Mg²⁺-free Krebs solution, MgSO₄ was replaced by Na₂SO₄. For electrophysiological measurements, the reference solution contained (in mmol/L) 148 sodium gluconate, 5 potassium gluconate, 2 NaCl, 1 MgCl₂, 1 CaCl₂, and 10 HEPES, brought to pH 7.4 with NaOH. The Mg²⁺-free solution contained (in mmol/L) 144.6 sodium gluconate, 5 K-gluconate, 5.4 NaCl, 0.3 CaCl₂, and 10 HEPES, brought to pH 7.4 with NaOH. The pipette solution used for both whole-cell and single channel (outside-out patches) contained (in mmol/L) 150 cesium gluconate, 3 MgCl₂, 5 EGTA, and 10 HEPES, brought to pH 7.2 with the addition of CsOH.

Statistics
All results are expressed as mean±SEM, with n being the sample size. Significance was tested by means of the Student t test. Probability values <5% (P0.05) were considered significant. Dose-response curves were fitted to a logistic equation using Origin software.

Chemicals and Drugs
Collagenase was from Worthington Biochemical Corp. Pronase (type E), elastase, BSA, noradrenaline, ATP, BzATP, ß-MeATP, oATP, ethidium bromide, ß-escin, diethylaminoethyl-dextran, and chloroquine were purchased from Sigma Chemical Co. 2-MeSATP was from Research Biochemicals International.

	Results

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Effect of ATP and Analogues on Tension in Human Saphenous Vein
Stimulation of denuded rings of human saphenous veins with ATP or 2-MeSATP (2 nonselective analogues of P2X receptors), ß-MeATP (agonist of P2X₁ and P2X₃ receptors), and BzATP (the better agonist of P2X₇ receptors) caused a concentration-dependent rise in tension. The contractions induced by 2-MeSATP were more transient than those evoked by the other P2X agonists (Figure 1A). The rank order of potency of the purinoceptor agonists defined from their EC₅₀ was ß-MeATP (7.2±1.4 µmol/L, n=9)>2-MeSATP (16.8±1.2 µmol/L, n=16)>BzATP (26.9±1.1 µmol/L, n=28)>ATP (182.0±1.4 µmol/L, n=7). Concentration-response curves to ATP and BzATP were modified by the removal of external Mg²⁺ (Figure 1B). The curves were shifted toward lower agonist concentrations, and the maximal tension was increased. The EC₅₀ for ATP and BzATP was decreased to 28.8±1.2 µmol/L (n=27, P<0.001) and 16.6±1.2 µmol/L (n=20, P<0.01), respectively. Maximal responses to 2-MeSATP were also increased similarly by the removal of Mg²⁺, but its EC₅₀ was not significantly changed (13.8±1.3 µmol/L, n=12, P>0.3; not shown). On the other hand, responses to ß-MeATP were not modified in Mg²⁺-free solution. Neither the maximal contraction nor the EC₅₀ was changed (8.3±1.2 µmol/L, n=14, P>0.7; Figure 1B).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effects of ATP and analogues on tension of human saphenous vein. A, Typical contractile response induced by increasing concentrations of ATP, 2-MeSATP, BzATP, or ß-MeATP. Agonist concentrations are indicated above the traces. B, Concentration-response curves to ATP (a), ß-MeATP (b), and BzATP (c) in the presence (1 mmol/L) (solid symbols) or absence (open symbols) of Mg2+ in Krebs-Henseleit solution. C, Concentration-response curves to ß-MeATP (a) and BzATP (b) under control conditions (open symbols) and after treatment with oATP (250 µmol/L, 2 hours; solid symbols). Tension was expressed as percentage of the response elicited by 10 µmol/L noradrenaline. Each point represents the mean±SEM of 7 to 27 experiments. *P<0.01.

oATP has been shown to irreversibly inhibit the ATP-induced permeabilization of macrophage and irreversibly block P2X₇ current after preincubation of 1 to 2 hours.^{11 16} The sensitivity of ATP analogue–induced tension to oATP was assessed by pretreating venous rings with oATP (250 µmol/L) for 2 hours. Figure 1C shows that the concentration-response curve to ß-MeATP after removal of oATP was not modified by preincubation with oATP (EC₅₀=8.9±2.8 µmol/L, n=8; P>0.7), whereas contractions induced by BzATP were strongly inhibited by oATP.

This suggests that the ATP-induced tension in saphenous veins might involve at least 2 different receptors: the first could be activated by ß-MeATP, insensitive to external Mg²⁺ and oATP; the second was sensitive to BzATP, external Mg²⁺, and oATP. These properties resembled those of P2X₁ receptor, already identified in other smooth muscle cells⁷ or P2X₃ receptor described in heart,¹⁴ and surprisingly those of P2X₇ receptor of macrophage,^{11 17} respectively.

Analysis of P2X Receptor mRNA in Smooth Muscle From Human Saphenous Vein
Reverse transcription and PCR amplification of human saphenous vein muscle total RNA with specific oligonucleotides indicated the presence of both hP2X₁ receptor and hP2X₇ receptor transcripts but the absence of hP2X₃ receptor transcript (Figure 2A). Figure 2B illustrates Northern blot analysis of total mRNA extracted from saphenous vein smooth muscle from 9 donors with hP2X₁ and hP2X₇ receptor–specific probes. Statistical analysis revealed that the hP2X₁/GAPDH signal ratio was 27.6±0.9 times higher than the hP2X₇/GAPDH signal ratio (n=9). Comparison of the nucleotide sequence of the PCR products with the corresponding regions of the human urinary bladder P2X₁⁷ and the human monocyte P2X₇¹¹ receptors revealed 100% and 99.85% identity, respectively. Similar results were obtained from healthy saphenous veins (not shown), indicating that expression of these receptors, in particular hP2X₇, is a feature of normal vascular myocytes rather than a marker of varicose veins. These results indicate that potentially both P2X₁ and P2X₇ receptors could be expressed in smooth muscle cells from human saphenous veins. A more detailed analysis of the ATP analogue–induced responses was therefore conducted by means of electrophysiological measurements in freshly isolated myocytes from human saphenous vein.

View larger version (74K): [in this window] [in a new window]   Figure 2. Expression of hP2X1, hP2X7, and hP2X3 receptor transcripts. A, RT-PCR was performed on RNA from human saphenous vein smooth muscle (hsv) with primers against the hP2X receptor subtypes indicated above. Gels are shown with contrasts inverted. RNA from HL60 cells and human newborn cardiac ventricle were used as positive controls. Negative controls (cont) were performed in the absence of RT. Also shown is a 1-kb DNA ladder (MWM). B, Expression of hP2X1 and hP2X7 was compared in RNA samples isolated from human saphenous vein. Northern blots were probed as indicated. GAPDH signal is used as a reference for loading. RNA from HL60 cells, U937 cells, and G2 hepatocytes were used as controls.

Properties of Whole-Cell Current Induced by ATP Analogues in Freshly Isolated Myocytes From Human Saphenous Veins
In Mg²⁺-free solution (0.3 mmol/L Ca²⁺), stimulation of freshly isolated venous myocytes clamped at a holding potential of -40 mV by BzATP induced an inward current consisting of a rapidly inactivating component followed by a maintained current (Figure 3A). This typical current was observed in 48 of 54 cells. The maintained component of the BzATP-activated current was strongly and reversibly reduced by increasing the concentration of divalent cations in the external solution (1 mmol/L Ca²⁺ and 1 mmol/L Mg²⁺). Under similar conditions, application of ß-MeATP (100 µmol/L) induced, with a short latency, a transient inward current (Figure 3B). The ß-MeATP–induced current was completely desensitized in the continuous presence of agonist with a half-decay time of 1.4±0.3 seconds (n=12). This current corresponded to the nonselective cation current previously characterized.¹³ After complete desensitization of the ß-MeATP–induced current, addition of BzATP (100 µmol/L) activated a maintained inward current only. The BzATP-activated current displayed no, or only small and slow, desensitization during BzATP application for a period as long as 3 minutes. In the continuous presence of ß-MeATP, identical currents could be repetitively activated by successive applications of BzATP (100 µmol/L, Figure 4A). The current-voltage relationship of the maintained BzATP-activated current recorded in the presence of ß-MeATP was approximately linear in the range of -120 to +40 mV with a reversal potential of +3.0±1.5 mV (n=6), suggesting that it involved a nonselective cation conductance (Figure 4B).

View larger version (17K): [in this window] [in a new window]   Figure 3. ß-MeATP– and BzATP–induced currents in freshly isolated human saphenous vein myocytes. A, In Mg2+-free solution at -40 mV, BzATP activated a transient inward current followed by a maintained component that was inhibited by increasing divalent cation concentrations. B, In cells maintained at a holding potential of -40 mV in Mg2+-free solution, ß-MeATP (100 µmol/L) rapidly induced an inward desensitizing current. Addition of BzATP (100 µmol/L) activated a maintained inward current. Voltage steps to +40, 0, -80, and -120 mV were applied in the absence and presence of agonists.

View larger version (16K): [in this window] [in a new window]   Figure 4. Properties of BzATP-induced current in freshly isolated venous myocytes. A, In Mg2+-free solution at a holding potential of -40 mV and in the continuous presence of ß-MeATP (100 µmol/L), successive applications of BzATP (100 µmol/L) repetitively evoked a maintained inward current without desensitization. To construct the current-voltage relationship, voltage steps to + 40, 0, -80, and -120 mV were applied before and during BzATP application. B, Current-voltage relationships for the BzATP-activated maintained current. Current amplitude was expressed as percentage of its amplitude at -120 mV. Each point represents the mean of 6 experiments.

Taken together, the results above indicate that in venous myocytes, P2X receptor agonists could activate 2 different types of cation currents: (1) a transient current, activated by ß-MeATP and BzATP, displaying desensitization in the presence of agonists and (2) a maintained current, insensitive to ß-MeATP, activated by BzATP and inhibited by external divalent cations. Thus, we aimed to analyze whether the properties of the P2X receptor cation currents recorded in human myocytes could be ascribed to the activation of hP2X₁ and hP2X₇ receptors or heteropolymers formed by assembly of hP2X₁ and hP2X₇ by functional expression of these receptors in COS cells.

Functional Expression of hP2X₁ and hP2X₇ Receptors in COS Cells
In COS cells expressing hP2X₁ receptors, ß-MeATP (10 µmol/L) or BzATP (100 µmol/L) activated a rapidly desensitizing current (Figure 5Aa). The half-decay time of the ß-MeATP–and BzATP-induced currents measured at a holding potential of -40 mV corresponded to 0.29±0.02 (n=10) and 0.29±0.02 seconds (n=11), respectively. However, in the continuous presence of ß-MeATP, after complete desensitization of the current, addition of BzATP (100 µmol/L) had no effect, indicating that BzATP was also an hP2X₁ agonist (Figure 5Ab). On the other hand, in cells transfected with hP2X₇ receptor, application of ß-MeATP (10 to 300 µmol/L) was without effect, whereas BzATP (30 µmol/L) activated a large current that was maintained until BzATP application was terminated (Figure 5B). Similar currents could be repetitively evoked by successive applications of BzATP. The current evoked by BzATP at hP2X₇ receptor was inhibited by increase of the external divalent cation concentration (not shown). Thus, both currents displayed properties similar to those described for the P2X₁ and P2X₇ receptors expressed in HEK293 cells.^{11 12}

View larger version (17K): [in this window] [in a new window]   Figure 5. Functional expression of hP2X1 and hP2X7 in COS cells. A, In COS cells expressing hP2X1 receptor, ß-MeATP (10 µmol/L) or BzATP (100 µmol/L) induced a transient inward current (a). In the continuous presence of ß-MeATP (10 µmol/L) after complete desensitization of the current, BzATP (100 µmol/L) had no effect (b). B, In COS cells expressing hP2X7 receptors, ß-MeATP (100 µmol/L) was completely ineffective, whereas BzATP (100 µmol/L) induced a large maintained current. C, In COS cells cotransfected with hP2X1 and hP2X7, ß-MeATP (10 µmol/L) induced a transient current. After complete desensitization in the continuous presence of ß-MeATP, addition of BzATP (100 µmol/L) induced a large maintained current. All traces were obtained in Mg2+-free solution at a holding potential of -40 mV and were representative of at least 10 cells for each condition.

Because mRNA for both hP2X₁ and hP2X₇ receptors was found in smooth muscle from human saphenous vein, we analyzed the properties of currents induced by hP2X receptor agonists in COS cells cotransfected with hP2X₁ and hP2X₇. Coexpression of hP2X₁ with hP2X₇ receptors led to fast-rising desensitizing ß-MeATP–induced currents similar to those recorded when hP2X₁ receptor was expressed alone (Figure 5C). In cotransfected cells, the half-decay time of the current induced by 10 µmol/L ß-MeATP at a holding potential of -40 mV was 0.29±0.02 seconds (n=14, P>0.5). In the continuous presence of ß-MeATP, addition of BzATP induced a slow nondesensitizing current, maintained until the agonist application was terminated, that could be repetitively evoked by successive BzATP applications and therefore was similar to the hP2X₇ receptor current recorded in cells expressing hP2X₇ receptor alone. These results suggest that in cotransfected COS cells, subunits hP2X₁ and hP2X₇ did not associate to form heteropolymers but behaved as independent populations of channels. However, hP2X receptor agonist–activated currents in cotransfected COS cells (Figure 5C) closely resembled those recorded in freshly isolated saphenous vein myocytes (Figure 3B), indicating that both hP2X₁ and hP2X₇ receptors were expressed as separate populations of ATP-gated channels, carrying the fast desensitizing and the maintained cation currents, respectively.

BzATP-Induced Formation of Nonselective Pores
The P2X₇ receptor has been reported to have dual functions, operating both as an ion channel selective for small cations and as a pore permeable to large molecules, leading to cell lysis.¹¹ The ability of the hP2X₇ receptor to induce pore formation was less than that of the rat P2X₇.¹⁷ The BzATP-induced formation of large pores depended on both external divalent cation concentration and temperature.¹⁸ We used ethidium bromide (394 Da), which increases its emission intensity when bound to nucleic acids, as a large molecular mass probe to detect by fluorescence measurements the formation of large nonselective pores in the presence of BzATP. At room temperature, BzATP did not induce pore formation in venous myocytes or in COS cells expressing hP2X₇ receptor (not shown). At 37°C, in Mg²⁺-free solution, BzATP (100 µmol/L) rapidly induced ethidium bromide uptake both in freshly isolated human myocytes and in COS cells expressing hP2X₇ receptor (Figure 6). After 10 minutes, the fluorescence intensity was increased to 19±3% (n=8) and 32±3% (n=11) of the ß-escin–induced rise in fluorescence in freshly isolated human myocytes and in COS cells expressing hP2X₇ receptor, respectively (Figure 6B). BzATP (100 µmol/L) did not induce change in fluorescence in COS cells expressing hP2X₁, indicating that the observed ethidium bromide uptake was related to the expression of hP2X₇ receptors.

View larger version (22K): [in this window] [in a new window]   Figure 6. Ethidium bromide uptake induced by BzATP (100 µmol/L) in COS cells expressing hP2X7 and in human saphenous vein myocytes. A, Typical trace illustrating the increase in ethidium bromide fluorescence induced by BzATP (100 µmol/L) and ß-escin (200 µmol/L) in COS cells expressing hP2X7 receptors. B, Time course of BzATP-induced rise in ethidium bromide fluorescence in COS cells expressing hP2X7 receptors (solid circles) and in freshly isolated human saphenous vein myocytes (open circles). BzATP (100 µmol/L) did not induce ethidium bromide uptake in COS cells expressing hP2X1 receptors (squares). All results were obtained at 37°C in Mg2+-free solution. Each point represents the mean±SEM of 8 to 11 experiments.

	Discussion

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The major finding of the present study is that the hP2X₇ receptor identified as the receptor involved in the ATP-induced cell lysis in immune/inflammatory cells was expressed in smooth muscle from human saphenous veins, in addition to the hP2X₁ receptor. We show that both hP2X₁ and hP2X₇ receptors were involved in the contractile effect of ATP and that activation of hP2X₇ could lead to the formation of membrane pores permeable to large molecules responsible for cell lysis.

Contribution of hP2X₁ and hP2X₇ Receptors to Contractile Effect of ATP
Our results indicate that both P2X₁ and P2X₇ found to be expressed in human saphenous vein myocytes contribute to the contractile effect of ATP.

The involvement of the hP2X₁ receptor arises directly from the dose-dependent contracting action of ß-MeATP and 2-MeSATP and is supported by the presence of (1) hP2X₁ mRNA in saphenous vein smooth muscle and (2) a rapidly desensitizing ß-MeATP–sensitive current in freshly isolated myocytes.

The contracting effect of the P2X₇ receptor agonist BzATP did not directly argue for the involvement of hP2X₇ receptor in the modulation of the venous tone because we demonstrated, using COS cells transfected with hP2X₁ cDNA, that BzATP was also an agonist of hP2X₁ receptors. However, the contracting action of the hP2X₇ receptor activation is supported by the following observations. (1) The contraction induced by BzATP and ATP was more maintained than that produced by 2-MeSATP. Because ATP, BzATP, and 2-MeSATP were all rapidly degraded by ecto-ATPases,¹⁹ this suggests that the difference in the time course of the contractions was due to the ability of ATP and BzATP but not 2-MeSATP to activate a maintained mechanism. (2) The ATP- and BzATP-induced contractions were inhibited by pretreatment with oATP, whereas the ß-MeATP–induced rise in tension was not affected. (3) The increase in the maximal response to ATP and BzATP and the decrease in EC₅₀ concentrations induced by the removal of external magnesium suggest a role of the tetrabasic form of the agonist (ATP^4-, BzATP^4-) known to be the active agonist of P2X₇ receptor.¹¹ (4) mRNA encoding for hP2X₇ receptor is present in saphenous vein smooth muscle. (5) In isolated myocytes, as in COS cells expressing hP2X₇ receptor, BzATP and ATP activated a maintained current insensitive to ß-MeATP.

It is therefore surprising to observe that ß-MeATP, which did not activate hP2X₇ receptor, produced a large and maintained contractile response and a cation current with a half-decay time approximately 5 times slower in myocytes than in transfected COS cells. This result could not be ascribed to a heteropolymer formed by the assembly of hP2X₁ and hP2X₇ subunits, conferring ß-MeATP sensitivity and a noninactivating property to the channel, respectively. Functional coexpression of hP2X receptors clearly demonstrated that such a heteropolymerization of hP2X₁ and hP2X₇ subunits did not occur. The absence of hP2X₃ receptor transcript in saphenous vein indicates that this other ß-MeATP–sensitive P2X receptor was not responsible for these results. The more potent contractile effect of ß-MeATP compared with that of ATP therefore could be due to the inhibition of ecto-ATPase activity.²⁰ By inhibiting ecto-ATPase activity, ß-MeATP slowed down its own degradation and could thus amplify hP2X₁ receptor activation. Such an effect could not be responsible for the maintained contractile effect of BzATP, since BzATP did not modify ecto-ATPase activity.²⁰ Another hypothesis would be that another isoform, not yet cloned of ß-MeATP–sensitive P2X receptors, was expressed in human venous myocytes.

In summary, our results suggest that activation of both hP2X₁ and hP2X₇ receptors expressed as separate sets of channels in human saphenous vein myocytes modulates venous tone.

Possible Role of P2X₇ Receptor Activation–Induced Membrane Pore Formation in Human Saphenous Vein
P2X₇, the larger receptor/channel of the P2X subfamily (595 amino acids), differs from other members of the P2X subfamily by the presence of a long cytoplasmic carboxy tail that is essential for the pore-forming activity, although not for its function as an ATP-activated channel.¹¹ For a long time, the only well-characterized and generally accepted effect consequent to P2X₇ stimulation has been cell death.²¹ Opening of the P2X₇ pore causes large transmembrane ion fluxes (Ca²⁺ and Na⁺ entry; K⁺ outflow) and loss of cytoplasmic low molecular weight metabolites. However, it was suggested very recently that the physiological function of P2X₇ receptors was unlikely to be cell death, but rather that it could be related to a transient nonlytic opening sufficient to allow passage of high molecular substance in and out of the cell.^{22 23} Cell lysis occurred only when the activation of P2X₇ receptor agonist was continuous for more than 60 seconds.

We found that activation of hP2X₇ receptors in both human venous myocytes and transfected COS cells could form nonselective membrane pores permeable to large molecules. This effect seems to require particular conditions (high agonist concentrations, maintained stimulation) and to strongly depend on temperature. Under physiological conditions, ATP concentration in the extracellular fluids is low (5 to 20 µmol/L),²⁴ and locally released ATP, in particular from endothelial cells,²⁵ is rapidly hydrolyzed by ecto-ATPase activity.^{26 27} Therefore, in physiological conditions, P2X₇ is expected to act, as do other P2X receptors, only as a cation channel allowing cation entry and membrane depolarization. Indeed, as described above, tension measurements suggest that hP2X₇ was involved in the contracting effect of ATP in human saphenous vein.

However, several observations could lead to the assumption that venous diseases offer conditions allowing the hP2X₇ receptor activation to cause lysis of venous myocytes. Several events may generate large localized ATP increases.²⁸ However, the most relevant source of ATP is the cytoplasm from which ATP can be released after hypoxia, stress, inflammation, or membrane damage, conditions that are found in the vessel wall of varicose veins.^{29 30 31} Maintained increase in external ATP concentration could also be generated by a reduced ecto-ATPase activity. Vascular smooth muscle cells displayed a potent ecto-ATPase activity depending on the presence of membrane ATP-diphosphohydrolase.³² This enzymatic activity is inhibited by inflammatory mediators (tumor necrosis factor-) and oxidant exposure.³³ It thus appears that the pathological conditions (stasis, hypoxia, inflammation) found in varicose veins favored the accumulation of high ATP concentration in the extracellular space. Therefore, it could be hypothesized that cell lysis consequent to hP2X₇ receptor activation contributes to the disorganization and decrease in the amount of contractile myocytes in the muscle layers of the media of varicose veins.³¹ The well-known deleterious effects of heat on venous disease support this hypothesis. Temperature of cutaneous veins is influenced by the external temperature, so a warm environment could favor the pore-forming activity of hP2X₇ receptors, shown to depend on temperature.

	Selected Abbreviations and Acronyms

 

ß-MeATP = ß-methylene-ATP BzATP = 2',3'-(4-benzoyl)benzoyl-ATP FCS = fetal calf serum oATP = oxidized ATP 2-MeSATP = 2-methylthioATP PSS = physiological saline solution RT-PCR = reverse transcription–polymerase chain reaction

	Acknowledgments

 
This work was supported by the CNRS and grants from the Fondation pour la Recherche Médicale and the Association Recherche et Partage. Gervaise Loirand and Annie Ladoux were supported by INSERM, and Pierre Pacaud was supported by CNRS. We gratefully acknowledge the assistance of Professor Batt and the members of the Service de Chirurgie Vasculaire, Hôpital Saint Roch, Nice, France, in supplying us with human saphenous samples. We thank Professor Coquerel from CHR, Rouen, France, for kindly providing us with samples of human newborn cardiac ventricle. We thank Dr Tedgui for helpful advice in the preparation of this manuscript.

Received February 10, 1998; accepted May 11, 1998.

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