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(Circulation Research. 1997;80:170-178.)
© 1997 American Heart Association, Inc.

Articles

Electrophysiological Consequences of Purinergic Receptor Stimulation in Isolated Rat Pulmonary Arterial Myocytes

S. Anthony Hartley, Roland Z. Kozlowski

the University Department of Pharmacology, Oxford, England.

Correspondence to Dr Roland Z. Kozlowski, University Department of Pharmacology, Mansfield Road, Oxford, OX1 3QT, England, UK. E-mail roland.kozlowski@pharm.ox.ac.uk

	Abstract

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Neither the electrophysiological effects of purinergic receptor stimulation nor the role of ATP in regulating the tone of pulmonary arterial smooth muscle has been determined. Therefore, we investigated the effects of purine nucleotides on acutely dissociated smooth muscle cells from rat small pulmonary arteries using the patch-clamp recording technique. Extracellular application of ATP activated a fast transient inward current (which decayed in the continued presence of the nucleotide) and produced sustained periodic oscillations of predominantly inward current. Pharmacological and anion substitution experiments revealed that the transient inward current was carried by the movement of cations. In contrast, the periodic oscillations of current were due primarily to a Ca²⁺-activated Cl^- current (I_Cl,Ca) dependent on the release of Ca²⁺ from intracellular stores. Experiments using ATP analogues revealed the following order of potency for activation of the fast transient inward current: 2-methylthio ATP (2-meSATP)>ATP>,ß-methylene ATP (,ß-meATP)>>ADP>UTP=adenosine. Cross desensitization was seen between applications of ATP, ,ß-meATP, and 2-meSATP, suggesting that these agonists act via a common site. The order of potency for activation of I_Cl,Ca was UTP=ATP>>ADP2-meSATP>,ß-meATP=adenosine. Both the fast transient inward current and I_Cl,Ca evoked by ATP and its analogues were abolished by the nonselective P₂ purinoceptor antagonist suramin. These results show the existence of P_2X and P_2U purinoceptor subtypes in pulmonary arterial smooth muscle cells. Stimulation of these receptors results in activation of a fast transient inward cation current and I_Cl,Ca, respectively. It is likely that ATP acts via these receptor subtypes to regulate pulmonary arterial tone under physiological or pathological conditions.

Key Words: pulmonary artery • P₂ purinoceptor • purine nucleotide • Cl^- channel

	Introduction

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In addition to its essential intracellular role in providing a molecular energy source, the purine nucleotide ATP also has potent and diverse extracellular actions on cardiovascular function and the regulation of blood flow, being released from the hypoxic myocardium,¹ endothelial cells,² damaged vessel walls,³ and aggregating platelets⁴ as well as from perivascular nerves, where it is known to act as an excitatory neurotransmitter.⁵

ATP mediates its various extracellular effects on cells by activating specific cell surface receptors, defined as P₂ purinoceptors, which have been subdivided into at least six subtypes, P_2X, P_2Y, P_2T, P_2U, P_2Z, and P_2D, largely on the basis of the relative agonist potencies of ATP and a number of its structural analogues, in particular ,ß-meATP and 2-meSATP.⁶ The cloning of the genes for the P_2Y and P_2U purinoceptors^{7 8} has shown that these receptors are members of the G protein–coupled receptor superfamily and are linked to various second messenger systems, such as phospholipase C,⁹ phospholipase A₂,¹⁰ phospholipase D,¹¹ and adenylate cyclase.¹² In contrast, the P_2X purinoceptor is coupled to a ligand-gated cation channel.¹³ Multiple subtypes of this receptor have recently been cloned. These have been defined as P_2X1, P_2X2, P_2X3, P_2X4, P_2X5, and P_2X6, and all have different patterns of expression as well as different pharmacological profiles.^{14 15 16 17 18 19}

In vascular smooth muscle, P_2X purinoceptors mediate vasoconstriction.⁶ In contrast, stimulation of P_2Y purinoceptors, typically located on the endothelium,²⁰ elicits vasodilatation at elevated levels of muscle tone. P_2U purinoceptors have been reported to be present either on the endothelium, where they mediate vasodilatation, or on smooth muscle, where they mediate vasoconstriction.²¹ In the pulmonary circulation, ATP has a dual effect on isolated vessels, causing contraction at resting tension and inducing relaxation of precontracted vessels.^{22 23} The contraction is mediated by P_2X purinoceptors located on smooth muscle, whereas relaxation is mediated by P_2Y purinoceptors located on the endothelium.²² Note, however, that P_2Y purinoceptors have also been identified on smooth muscle of human small pulmonary arteries,²³ a finding that highlights considerable species-dependent variation in purinoceptor distribution.^{24 25}

ATP has been reported to partially inhibit HPV²⁶ and reduce pulmonary vascular resistance in human patients with chronic obstructive pulmonary disease and pulmonary hypertension.²⁷ Indeed, ATP may serve to dilate the pulmonary circulation through its release from endothelial cells as a consequence of shear stress.²⁸ Alternatively, ATP may constrict pulmonary arteries by being released as an excitatory cotransmitter with noradrenaline at sympathetic nerve terminals²⁹ or from purinergic (nonadrenergic) excitatory nerves.³⁰ In order to help elucidate the mechanisms underlying purinergic control of pulmonary arterial smooth muscle, we have examined the electrophysiological actions of ATP and some of its structural analogues on single smooth muscle cells acutely dissociated from rat small pulmonary arteries. Results from the present study clearly show the existence of two types of purinergic receptor that upon stimulation produce markedly different electrophysiological responses that are likely to contribute to the control of pulmonary vascular tone.

	Materials and Methods

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Single-Cell Isolation
Male albino Wistar rats (200 to 250 g body weight) were killed by an overdose of intraperitoneal Euthatol (pentobarbitone sodium British Pharmacopoeia, Rhone Merieux) and cervically dislocated. Small pulmonary arterial vessels, 200 to 400 µm in diameter, were removed from the lungs, and smooth muscle cells were isolated using a dispersion procedure (previously described by Albarwani et al³¹ ) modified to include an incubation with collagenase (type VIII, 1.5 mg/mL), protease (type I, 0.1 mg/mL), and elastase (type II-A, 0.3 mg/mL) for 4 minutes at 37°C after pretreatment with papain. Cells were stored at 4°C and remained viable for 10 hours.

Electrophysiological Recordings
Membrane current from pulmonary arterial myocytes was recorded using either the perforated-patch³² or whole-cell³³ configuration of the patch-clamp recording technique. The former configuration was used in both current- and voltage-clamp modes. To examine the effects of purinergic receptor stimulation on membrane current, cells were voltage-clamped at -50 mV unless otherwise stated. Electrodes were pulled from borosilicate glass capillaries (Clark Electromedical) using a vertical puller (Narishige Ltd). Ionic currents were detected using an Axopatch 200A amplifier (Axon Instruments). Series resistance and capacity compensation facilities were used where necessary. Data were filtered at 2 kHz using a Digidata 1200 interface (Axon Instruments) and recorded, either on-line with a personal computer or off-line with a modified DAT recorder (Sony DTC-100ES). Data were analyzed using pClamp software (version 6.02, Axon Instruments Inc).

Solutions
The composition of the solutions used throughout the present study is given in the Table. For the majority of electrophysiological recordings, pulmonary arterial myocytes were bathed in a quasiphysiological solution in the presence (solution A) or absence (solution B) of Ca²⁺. For perforated-patch recordings, the pipette contained solution C, to which 240 µg/mL amphotericin B was added. In order to investigate the effects of changes in [Ca²⁺]_i on membrane current, cells were dialyzed in the whole-cell configuration with solution D, which contained a free [Ca²⁺] of 1 nmol/L. These values of free Ca²⁺ were calculated using a computer program based on the equations described by Fabiato.³⁴ To examine the ionic basis of currents activated as a consequence of purinergic receptor stimulation, cells were maintained in different Cl^- gradients, either bathed in solution E while solution F was contained within the pipette or bathed in solution G while solution H was contained within the pipette. More detailed anionic substitution experiments were carried out using solution I in the bath and solution H in the pipette. In this series of experiments, 120 mmol/L Cl^- was replaced with either I^- or Br^-. Similarly, a series of more detailed cationic substitution experiments was performed. For these experiments, solution J (in the absence or presence of 2 mmol/L Ca²⁺) and solution K were used to bathe the cells, while solution C dialyzed the cell interior. All experiments were performed at room temperature (20°C to 24°C). Agonists were applied to the bath using a gravity feed system, and the time for complete solution exchange was 4 s.

View this table: [in this window] [in a new window]   Table 1. Composition of Internal and External Solutions

Drugs
Adenosine, ATP (disodium salt), ,ß-meATP (lithium salt), amphotericin B, caffeine, collagenase (type VIII), dithiothreitol, elastase (type II-A), EGTA, GTP (sodium salt), iberiotoxin, HEPES, niflumic acid, papain (papaya latex), protease (type I), and UTP (sodium salt) were all purchased from Sigma Chemical Co. Manoalide and suramin (hexasodium salt) were obtained from ICN Biochemicals Ltd, and 2-meSATP (tetrasodium salt) was obtained from Research Biochemicals Inc.

Data Analysis
Data are presented throughout as mean±SEM. When presented graphically, the SEM is represented by the associated error bars. Statistical significance was assessed using Student's t test. Values of P.05 were considered significant. In order to compare the ability of a range of ATP analogues to activate oscillatory inward currents, membrane current elicited over a 1-minute period was digitized at 100 Hz and integrated (using pClamp software, version 6.02), as previously described,³⁵ giving a value in nanoamperesxmilliseconds.

	Results

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Electrophysiological Effects of ATP
Single cells isolated from rat small pulmonary arteries had a resting membrane potential ranging between -37.4 and -58.4 mV (-47.2±1.6 mV, n=16). ATP (10 µmol/L), when added to pulmonary arterial myocytes maintained in the perforated-patch configuration under current clamp (solution A in the bath and solution C in the pipette throughout), caused a rapid transient depolarization followed by oscillations in the membrane potential (Fig 1A). The potential importance of these changes in membrane potential in mediating contraction of pulmonary arterial smooth muscle prompted further investigation into their ionic basis. Consistent with current-clamp experiments, extracellular application of ATP (10 µmol/L throughout) to cells that were under voltage-clamp mode and maintained in the permeabilized-patch configuration (solution A in the bath and solution C in the pipette) evoked two different inward currents at a holding potential of -50 mV (close to the resting membrane potential of pulmonary arterial myocytes³⁶ ). The first current was a fast transient inward current that lasted between 1 and 3 s (mean duration, 2.6±0.3 s; n=22) and decayed rapidly in the continued presence of the nucleotide (=467.3±72.3 ms, n=20). Although this desensitization (decay) developed in seconds, recovery required several minutes to occur after removal of the ATP. A subsequent application of the nucleotide (after 10 to 12 minutes) produced a similar response. The second current took the form of periodic oscillations that continued throughout the application of the nucleotide, generally applied for 1 to 3 minutes (Fig 1B). Note that after prolonged (6-minute) application of ATP, some rundown of the oscillations was observed.

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of extracellular application of ATP to pulmonary arterial myocytes. Cells were maintained in the perforated-patch recording configuration (solution A in the bath and solution C in the pipette) and exposed to ATP (10 µmol/L throughout) for the period indicated by the bars. A, Current-clamp recording of cell membrane potential (Vm). Before application of ATP, the resting Vm of this cell was -56 mV. After the addition of ATP, there was a rapid transient depolarization (*) followed by oscillations in Vm. B, Current recording obtained under voltage-clamp mode at a holding potential of -50 mV. ATP activated a fast transient inward current (*, which decayed in the continued presence of the nucleotide) and sustained periodic oscillations of inward current. Note that on occasion W-shaped current oscillations were observed (shown on a faster time base in the inset). C, Current recording showing the effects of ATP in the presence of iberiotoxin (20 nmol/L), a known selective blocker of KCa channels, on a cell voltage-clamped at -50 mV. Under these conditions, W-shaped current oscillations were not observed upon application of ATP (shown on a faster time base in the inset).

The transient inward current, seen in 51 of 59 cells, upon application of ATP reached a mean peak amplitude of 208.3±20.0 pA (n=22). Its onset time was 1.24±0.25 s (n=18). In contrast, a longer onset time of 11.5±2.6 s (n=22) was required before periodic oscillations of inward current, seen in 56 of 73 cells, were observed. The magnitude of this current, measured over a period of 1 minute, was 565.8±103.4 nA·ms (n=19). Note that although both the transient inward and the oscillatory inward currents were observed in the majority of cells, not all the data could be quantified. This was due to either a short experimental life time or contamination of the oscillatory inward current by simultaneous activation of an additional current component of varying magnitude, which imparted a "W-shaped" profile on an individual oscillation. Such effects were seen in 37 of 56 cells (see Fig 1B). This current profile (which appeared to contain an initial component resembling the transient inward current) was blocked in 7 of 8 cells by coapplication of 20 nmol/L iberiotoxin, a selective K_Ca channel blocker³⁷ (compare Fig 1B and inset with Fig 1C and inset). This indicated that the W-shaped current profile was due to activation of K_Ca channels and not to activation of the transient inward current (which was unaffected by iberiotoxin, Fig 1C).

Ionic Basis of Currents Activated by ATP
In order to determine the ionic basis of the inward currents activated by ATP, a number of anion substitution experiments were performed. Cells were maintained in the perforated-patch configuration under a Cl^- gradient of 155 mmol/L [Cl^-]_o (solution E, bath) and 17 mmol/L [Cl^-]_i (solution F, pipette), where the Cl^- reversal potential was -60 mV. Under this Cl^- gradient and at a holding potential of -30 mV, application of ATP still activated the transient inward current, but the current oscillations were in an outward direction (n=5; for example, see Fig 2A). This observation clearly indicates that the two current types are due to the movement of different ions. Given the ionic gradient used, it seemed likely that the transient inward current was carried by cations and that the oscillatory current was due to movement of either Cl^- or K⁺ ions. Further anion substitution experiments were performed to verify this possibility. Thus, cells were maintained in a Cl^- gradient of 143 mmol/L [Cl^-]_o (solution G, bath) and 42 mmol/L [Cl]_i (solution H, pipette), where the Cl^- reversal potential was -31 mV. Under these conditions at a holding potential of 0 mV (the approximate reversal potential for cations), application of ATP failed to activate the transient inward current but induced periodic oscillations of outward current (n=5). Upon changing the holding potential to -31 mV, the current oscillations disappeared (Fig 2B), indicating that the transient current is probably due to the inward movement of cations and that the current oscillations are due to the movement of Cl^- ions. Consistent with this observation, niflumic acid (50 µmol/L, a blocker of Cl^- channels,³⁸ ) reversibly inhibited oscillations of inward current in response to ATP application (n=10; for example, see Fig 2C) but failed to block activation of the transient inward current upon coapplication of ATP (n=5, data not shown). Additional detailed anion and cation substitution experiments were used to further characterize the nature of the ionic conductances activated by ATP. Upon replacing a proportion of the extracellular Cl^- with I^- or Br^- (solution I), the magnitude of the current oscillations was enhanced, with the relative permeability of these three anions being I^->Br^->Cl^- (Fig 3A). Note that these experiments were performed in the presence of iberiotoxin (20 nmol/L) to prevent activation of K_Ca channels and potential contamination of I_Cl,Ca. Substitution of extracellular cations with the impermeant cation choline (solution J) abolished the transient inward current, which remained unaffected by replacement of extracellular Na⁺ with K⁺ (solution K). In the absence of any monovalent cations, but in the presence of 2 mmol/L Ca²⁺ extracellularly, a small transient inward current was observed. These cation substitution data are illustrated quantitatively in Fig 3B. Taken together, results of these ion substitution experiments show quite clearly that the transient inward current flows through nonselective cation channels and that the current oscillations are due to the opening of Cl^- channels. Note that these ion substitution experiments also confirmed that the transient inward current did not contribute to the W-shaped currents (described above), since they were still observed in the presence of choline. Furthermore, under conditions in which the Cl^- current oscillations were outward (Fig 2A), the transient current was inward.

View larger version (20K): [in this window] [in a new window]   Figure 2. Ionic basis of the currents activated by ATP. Cells were maintained in the perforated-patch configuration and exposed to ATP (10 µmol/L throughout) for the period indicated by the bars. A, Current recording obtained from a cell voltage-clamped at -30 mV under a Cl- gradient of 155 mmol/L [Cl-]o (solution E, bath) and 17 mmol/L [Cl-]i (solution F, pipette). ATP activated a transient inward current and an oscillatory outward current. B, Current recording obtained from a cell voltage-clamped at 0 mV under a Cl- gradient of 143 mmol/L [Cl-]o (solution G, bath) and 42 mmol/L [Cl]i (solution H, pipette). Under these conditions, ATP failed to activate a transient inward current but activated an oscillatory outward current, which was abolished at -31 mV, the theoretical reversal potential of Cl- ions under this Cl- gradient. C, Current recording showing the effect of niflumic acid (50 µmol/L) applied extracellularly to a cell voltage-clamped at a potential of -50 mV.

View larger version (37K): [in this window] [in a new window]   Figure 3. Further evaluation of the ionic basis of ATP-activated currents. A, Bar graph showing the mean±SEM activation (currents measured in nA·ms; see "Materials and Methods") of ICl,Ca by ATP (10 µmol/L) at a holding potential of 0 mV with different halides in the extracellular solution (solution I). Solution H was contained within the pipette. Upon replacing a proportion of the extracellular Cl- (120 mmol/L) with I- or Br-, the magnitude of the current oscillations was enhanced, with the relative permeability of these three anions being I->Br->Cl-. Note that these experiments were performed in the presence of iberiotoxin (20 nmol/L) to prevent activation of KCa channels and potential contamination of ICl,Ca. B, Bar graph showing the mean±SEM activation (measured as the peak response in pA) of the fast transient inward current activated by ATP (10 µmol/L) in the presence of four different extracellular cationic environments (solution B, J, or K bathing the cells with solution C in the pipette) at a holding potential of -50 mV. Note that there is no difference between the magnitude of the current when extracellular Na+ is replaced with K+ and that the current is totally abolished in the presence of extracellular choline. It is also clear that Ca2+ can also pass through the channel, since a small current is apparent in the presence of choline and 2 mmol/L Ca2+ extracellularly. Throughout this figure the number of cells is shown in parentheses.

Role of Ca²⁺ in the Current Responses to ATP
Since the ionic substitution experiments described above did not obviate the involvement of extracellular Ca²⁺ in activating or contributing to either the transient or oscillatory inward currents, the effects of ATP were examined in the absence of the cation (solution B, bath). Under these conditions, the amplitude of the transient inward current (206.1±64.2 pA, n=7) was similar to control (208.3±20.0 pA, n=22). Likewise, the magnitude of the oscillatory current (315.0±100.6 nA·ms, n=7) in Ca²⁺-free conditions was similar to that observed (371.5±102.6 nA·ms, n=7) in the presence of 1.2 mmol/L extracellular Ca²⁺.

In order to determine whether intracellular Ca²⁺ was involved in activating either the transient or the oscillatory inward current, the effects of caffeine known to deplete intracellular Ca²⁺ stores were examined. Application of 10 mmol/L caffeine in Ca²⁺-free solution evoked short-lived but complex fluctuations in current (Fig 4A). Subsequent application of ATP (in the continued presence of caffeine) activated only the transient inward current (n=6), an observation that indicated that the oscillatory inward current was probably activated by Ca²⁺ released from intracellular stores (Fig 4A). The amplitude of the transient inward current activated by ATP in the presence of caffeine (10 mmol/L) was not significantly different (199.8±32.6 pA, n=6) from the response under control conditions (208.3±20.0 pA, n=22), confirming that it occurred independent of Ca²⁺ release from intracellular Ca²⁺ stores. Consistent with this notion, upon dialyzing of cells with a solution containing the Ca²⁺-chelating agent EGTA (solution D, pipette), application of ATP activated the transient inward current (peak amplitude, 211.4±67.8 pA; n=3) but not the oscillatory inward current (Fig 4B). In order to determine whether IP₃ was involved in mediating Ca²⁺ release from intracellular stores, the effects of manoalide, a phospholipase C inhibitor,³⁹ were examined. Three-minute pretreatment of the cells with manoalide (3 µmol/L) markedly reduced inward current oscillations in response to ATP, without affecting the transient inward current (Fig 4C). In this series of experiments, the current density of the inward current oscillations was 135±90 nA·ms (n=6) in the presence of manoalide compared with 565±103 nA·ms (n=29) under control conditions (P.01).

View larger version (16K): [in this window] [in a new window]   Figure 4. Role of intracellular Ca2+ in the current responses to ATP. Agents were applied for the period indicated by the bars. A, Current recording obtained using the perforated-patch recording configuration in the absence of extracellular Ca2+ (solution B in the bath and solution C in the pipette) at a holding potential of -50 mV. Application of caffeine (10 mmol/L) evoked short-lived but complex fluctuations in current. Subsequent application of ATP (10 µmol/L) activated a fast transient inward current but not periodic oscillations of inward current. B, Current recording obtained during whole-cell recording configuration under conditions in which the [Ca2+]i was buffered at 1 nmol/L (solution A in the bath and solution D in the pipette). Under these conditions and at a holding potential of -50 mV, application of ATP (10 µmol/L) activated a fast transient inward current but did not activate periodic oscillations of inward current. C, Current recording obtained using the perforated-patch recording configuration (solution A in the bath and solution C in the pipette) at a holding potential of -50 mV. The cell was pretreated with manoalide (3 µmol/L) before the concomitant application of ATP (10 µmol/L). Manoalide markedly reduced inward current oscillations in response to ATP, without affecting the transient inward current.

Bringing these results together with those from the previous section, it seems clear that I_Cl,Ca is responsible for the inward current oscillations seen in the presence of ATP.

Purine Receptors Underlying Activation of the Transient Inward Current
,ß-meATP is a methylated analogue of ATP and is a potent selective agonist at P_2X purinoceptor subtypes. Application of 10 µmol/L ,ß-meATP to pulmonary arterial myocytes maintained in the perforated-patch configuration at a holding potential of -50 mV evoked a transient inward current, which decayed in the continued presence of the agonist, having a similar time course of desensitization (=500.2±108.9 ms, n=4) to ATP (=467.3±72.3 ms, n=20) although the mean peak amplitude of the current was smaller. Under identical conditions, 10 µmol/L 2-meSATP also activated a fast transient inward current, although its mean peak amplitude was larger than that of either ATP or ,ß-meATP, and in all cells tested, a rapidly desensitizing component of the response was observed in addition to a slow component (_fast=75.5±15.5 ms and _slow=424.1±135.0 ms, n=5). ADP (10 µmol/L) also activated the transient inward current, although its potency was much reduced and desensitization prolonged when compared with the other agonists. Adenosine (10 µmol/L, n=4) and UTP (10 µmol/L, n=10) were ineffective at activating the fast transient inward current. In cells in which these agents were tested, subsequent application of ATP (10 µmol/L) evoked the fast transient current, confirming their viability. The relative potency for activating the fast transient current was 2-meSATP>ATP>,ß-meATP>ADP>UTP=adenosine, at a concentration of 10 µmol/L. Examples of these effects, together with a quantitative analysis, are shown in Fig 5A and 5B. Upon repeated applications of ATP (10 µmol/L), there was a marked reduction in the peak current amplitude of the fast transient inward current (note that such desensitization did not affect activation of I_Cl,Ca). Application of ATP (10 µmol/L) immediately after 10 µmol/L 2-meSATP (n=7) or 10 µmol/L ,ß-meATP (n=3) rendered it ineffective at initiating the fast transient inward current (Fig 5C). This cross desensitization suggests that ATP, 2-meSATP, and ,ß-meATP all evoke the transient inward current through an interaction with a common site.

View larger version (30K): [in this window] [in a new window]   Figure 5. Pharmacological basis of the transient inward current. Agents were applied for the period indicated by the bars. A, Current recordings obtained using the perforated-patch recording configuration (solution A in the bath and solution B in the pipette). ATP (10 µmol/L), ,ß-meATP (10 µmol/L), and 2-meSATP (10 µmol/L) all evoked fast transient inward currents when applied to cells at a holding potential of -50 mV. B, Bar graph showing the mean±SEM activation (measured as the peak response in pA) of the fast transient inward current by a number of purine nucleotides (10 µmol/L). The number of cells is shown in parentheses. C, Current recording obtained using the whole-cell recording configuration under conditions in which the [Ca2+]i was buffered at 1 nmol/L with EGTA (solution A in the bath and solution D in the pipette). Under these conditions and at a holding potential of -50 mV, application of ,ß-meATP (10 µmol/L) activates a fast transient inward current but prevents activation of a similar current by a subsequent application of ATP (10 µmol/L). D-i, Current recordings obtained using the perforated-patch recording configuration at a holding potential of -50 mV. The left trace shows that after 5 minutes of bath perfusion with suramin (100 µmol/L), concomitant application of ,ß-meATP (10 µmol/L) has no effect on the holding current. The right trace shows that 10 minutes after washout of suramin, an inward current can be evoked after reapplication of ,ß-meATP (10 µmol/L) in the same cell. D-ii, Bar graph showing mean±SEM inhibition of the fast transient inward currents activated by 2-meSATP, ATP, and ,ß-meATP (all 10 µmol/L) by suramin (100 µmol/L). The number of cells is shown in parentheses. The open bars represent the responses to ATP in the presence of suramin (100 µmol/L); the filled bars represent responses to ATP following suramin washout (after 10 minutes). *Significant (P.01) inhibition by suramin.

In a number of vascular and neuronal preparations, suramin has been shown to be an nonselective antagonist at P₂ purinoceptors.⁴⁰ To investigate the action of suramin on ATP-induced activation of the fast transient inward current, the effects of ATP, ,ß-meATP, and 2-meSATP were determined first in the presence and then, 10 minutes later, in the absence of the antagonist. This methodology was adopted so as to allow the antagonistic effect of suramin to be discriminated from any desensitization observed due to a second application of agonist. After application of suramin (100 µmol/L), coapplication of either ATP, ,ß-meATP, or 2-meSATP (all at 10 µmol/L) evoked a small inward current. After washout (10 minutes) of suramin, these three agonists all evoked large, fast, transient inward currents. These results are illustrated and quantified in Fig 5D.

Purine Receptors Underlying Activation of the Oscillatory Cl^- Current
Upon testing a range of purinergic agonists at a concentration of 10 µmol/L, UTP was found to evoke periodic oscillations of I_Cl,Ca in 14 of 17 cells with a potency similar to that for ATP (Fig 6A); ADP and 2-meSATP only weakly activated I_Cl,Ca in 1 of 5 and 1 of 6 cells, respectively; and both ,ß-meATP (n=8) and adenosine (n=4) were ineffective. The relative potency for activating I_Cl,Ca was UTP=ATP>ADP>2-meSATP>,ß-meATP=adenosine (Fig 6B). Application of 100 µmol/L suramin inhibited oscillations of I_Cl,Ca evoked by 10 µmol/L ATP or UTP (Fig 6C and 6D).

View larger version (36K): [in this window] [in a new window]   Figure 6. Pharmacological basis of the oscillatory Cl- current. Agents were applied for the period indicated by the bars. A, Current recordings obtained using the perforated-patch recording configuration (solution A in the bath and solution B in the pipette) at a holding potential of -50 mV. Application of UTP activated periodic oscillations of inward current but failed to activate the fast transient inward current. B, Bar graph showing the mean±SEM activation (currents measured in nA·ms; see "Materials and Methods") of ICl,Ca by a number of purine nucleotides (10 µmol/L). The number of cells is shown in parentheses. C, Current recording showing the inhibiting effect of extracellularly applied suramin (100 µmol/L) on ICl,Ca to a cell voltage-clamped at a potential of -50 mV. D, Bar graph showing the mean±SEM inhibition by suramin (100 µmol/L) of ICl,Ca activated by ATP and UTP (both 10 µmol/L). The number of cells is shown in parentheses. *Significant (P.01) inhibition by suramin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Electrophysiological Consequences of Purinergic Receptor Stimulation
In the present study, we have examined the electrophysiological effects of ATP and a number of its structural analogues on single smooth muscle cells isolated from rat small pulmonary arteries. Results from the present study reveal that application of ATP activates two distinct types of inward current: a fast transient cation current that rapidly desensitizes in the continued presence of the nucleotide and sustained oscillations of current that are predominantly due to the opening of Cl^- channels. This is the first report describing in detail the electrophysiological consequences of purinergic receptor stimulation in pulmonary arterial smooth muscle.

Receptor Characterization
Pharmacological experiments using a range of ATP analogues revealed that the transient inward current was due to the stimulation of P_2X purinoceptors and that the oscillations of inward current were due to the stimulation of P_2U purinoceptors. Agonists elicited a P_2X-mediated response (activation of the transient inward current) with the following order of potency: 2-meSATP>ATP>,ß-meATP>>ADP>UTP=adenosine. This order does not correspond to that described for P_2X purinoceptor–mediated vasoconstriction of isolated rat main²² and human small²³ pulmonary arteries (,ß-meATP>>2-meSATPATP). It is now widely recognized, however, that a weakness of characterizing P_2X purinoceptors in whole tissues is that the potency of some agonists may be greatly influenced by their susceptibility to degradation by ectonucleotidases. Indeed, in the absence of any agonist breakdown, either through experiments performed on single acutely dissociated smooth muscle cells⁴¹ or by using an ecto-ATPase inhibitor,⁴² the relative potency of ATP and its analogues at activating P_2X purinoceptors is consistent with the findings of the present study.

With respect to the current oscillations, the following order of potency was observed: UTP=ATP>>ADP 2-meSATP>,ß-meATP=adenosine. Of key importance is that UTP was found to be equipotent to ATP, with other purinergic agonists being notably less effective. This order of potency is indicative of the existence of P_2U purinoceptors.⁴³ These receptors are known to be present on smooth muscle, where they mediate vasoconstriction, or on the endothelium, where they mediate vasodilation.²¹ Our experiments did not reveal the presence of P_2Y purinoceptors. This is perhaps not surprising, since the purinoceptors are generally located on the endothelium^{20 22} (although they have been found to exist on the smooth muscle of human small pulmonary arteries²³ ). It is not known whether this difference in P_2Y purinoceptor localization reflects a species-dependent variation.

Although we have characterized the pharmacology of the purinergic receptors using a range of receptor agonists and antagonists, it is important to note that these receptors can be subdivided further at the molecular level. Indeed, the cloning of the genes for the P_2Y and P_2U purinoceptors has revealed the existence of a number of subtypes (for a review, see Abbracchio and Burnstock⁴⁴ ). Similarly, in the case of the P_2X purinoceptor, multiple subtypes defined as P_2X1, P_2X2, P_2X3, P_2X4, P_2X5, and P_2X6 have been identified. These have different patterns of expression as well as phenotypic differences, such as differential rates of desensitization and sensitivity to ,ß-meATP.^{14 15 16 17 18 19} It can be difficult to accurately discriminate these receptor subtypes pharmacologically, since in some tissues P_2X purinoceptor RNAs have a widespread and extensively overlapping distribution.¹⁸ In addition, some native receptors are known to be heteropolymers of different cloned subunits.⁴⁵ However, Collo et al¹⁸ have shown that the smooth muscle of some peripheral arteries only expresses RNA for the P_2X1 purinoceptor subtype, indicating that the P_2X response seen in the pulmonary artery can possibly be attributed to the existence of P_2X1 purinoceptors. Further molecular studies are therefore required to verify exactly the nature of the P_2X receptor identified in the present study.

Mechanisms Underlying the Electrophysiological Effects of P_2X and P_2U Purinoceptor Stimulation
On the basis of our experiments, the electrophysiological effects of P_2U purinoceptor stimulation are likely to be dependent on Ca²⁺ released from intracellular stores, since they (1) were observed in the absence of extracellular Ca²⁺, (2) were inhibited by extracellularly applied caffeine, and (3) were inhibited by manoalide. To further support this notion, electrophysiological responses following P_2U purinoceptor stimulation occurred only when using the perforated-patch configuration (which allows endogenous Ca²⁺ homeostatic mechanisms to function normally³² ) and were prevented by clamping [Ca²⁺]_i at a low level using EGTA during whole-cell recordings. These findings are in agreement with other studies showing that P_2U purinoceptor stimulation can increase IP₃ levels (for review, see Boarder et al⁴⁶ ), causing mobilization of intracellular Ca²⁺ resulting in the activation of I_Cl,Ca. However, they highlight marked differences in the electrophysiological effects of purinoceptor stimulation in a range of muscle types. For example, ATP activates a nonoscillatory Ca²⁺-activated Cl^- current in myocytes from pig aorta⁴⁷ but fails to activate such a current in rabbit portal vein⁴⁸ or ear artery.⁴⁹ Interestingly, however, in rat ventricular myocytes, purinoceptor stimulation has been reported to initiate oscillations of intracellular Ca²⁺,⁵⁰ although the electrophysiological effects have yet to be examined. At present, we cannot speculate as to the mechanisms underlying these tissue differences. However, they may be due to (1) different experimental procedures, (2) differential purinoceptor expression and coupling, or (3) subtle, as yet uncharacterized, differences in the Ca²⁺ homeostatic mechanisms present in these tissues. Although it is clear that in pulmonary arterial myocytes P_2U purinoceptor stimulation results in the release of Ca²⁺ from intracellular stores (likely to be IP₃ sensitive), the mechanism underlying Ca²⁺-induced activation of Cl^- (and K⁺ channels) was not investigated in the present study. However, it is likely that these effects are a consequence of Ca²⁺ binding directly to the channels rather than through indirect pathways involving, for example, Ca²⁺-dependent phosphorylation. Indeed, Ca²⁺-activated channels, activated by agonists, are known to exist in the pulmonary circulation.^{35 51}

Activation of the transient inward current as a consequence of P_2X purinoceptor stimulation is known to occur independent of phosphorylation or elevation of [Ca²⁺]_i,⁵² since molecular studies have revealed that the P_2X purinoceptor forms part of a ligand-gated ion channel.^{14 15} These findings are in agreement with results from the present study, since the current shows a rapid time constant of activation and activates independent of the presence of intracellular Ca²⁺ and under conditions unable to support phosphorylation (cell dialysis with solution C).

Physiological Role of Purinergic Receptor Stimulation
ATP, through stimulation of purinoceptors, causes constriction of the pulmonary circulation, which is a low-pressure low-resistance circuit that has little or no resting tone.²⁴ The P_2X and P_2U purinoceptors characterized in the present study were located on arterial smooth muscle cells. They can therefore be stimulated by ATP released (1) from nerve terminals innervating the smooth muscle,^{5 30} (2) from the smooth muscle itself,⁵³ or (3) from the endothelium.² In contrast, the effects of circulating ATP would be expected to be dependent on endothelial purinoceptors and the degree of muscular tone. Stimulation of endothelial P_2Y and P_2U purinoceptors mediates vasodilation via the release of NO or prostacyclin (for review, see Boarder et al⁴⁶ ) but only at elevated levels of tone.^{22 23} Thus, it is likely that the effect of ATP in the pulmonary circulation depends on the site of its release and the levels of arterial tone. Therefore, during HPV, ATP could be expected to relax the pulmonary circulation²⁶ (although luminal and abluminal endothelial release of ATP could be expected to have a differential action—relaxation and constriction, respectively). The electrophysiolgical effects described in the present study are consistent with a constricting action of ATP, since our experiments revealed inward (depolarizing) currents at -50 mV (the approximate resting membrane potential of these cells³⁶ ), and it is well known that depolarization is associated with constriction of smooth muscle.

To summarize, ATP elicits marked electrophysiological effects in pulmonary arterial smooth muscle cells (mediated through P_2X and P_2U purinoceptor stimulation), which are likely to be involved in inducing vasoconstriction, a potentially important physiological response. Indeed, ATP is known to be released from endothelial cells after sheer stress and hypoxia in a range of tissue types.⁵⁴ However, it remains to be determined whether such effects are applicable to the pulmonary circulation and whether they play a role in HPV. It is also possible that ATP may play a pathological role. For example, in certain forms of pulmonary hypertension, where there is loss or trauma to the endothelium, the release of ATP may contribute to vasoconstriction and, thus, to the elevation of arterial perfusion pressure.

	Selected Abbreviations and Acronyms

 

,ß-meATP = ,ß-methylene ATP HPV = hypoxic pulmonary vasoconstriction ICl,Ca = Ca2+-activated Cl- current IP3 = inositol tri-phosphate KCa channel = Ca2+-activated K+ channel 2-meSATP = 2-methylthio ATP = time constant

	Acknowledgments

 
This study was supported by Glaxo Wellcome. Dr Kozlowski is a British Heart Foundation lecturer.

Received July 1, 1996; accepted October 29, 1996.

	References

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