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MS 9813 Received 7 July 1999; accepted after revision 2 September 1999.
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ABSTRACT |
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INTRODUCTION |
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It is widely recognized that perivascular nerves and endothelial cells control the tone of the smooth muscle of vessel walls and hence regulate the local blood flow (Furchgott & Zawadzki, 1980; Burnstock, 1990). There is now considerable evidence indicating that ATP is a transmitter in the rabbit saphenous and mesenteric (Burnstock, 1990) and guinea-pig mesenteric (Onaka et al. 1997) arteries. ATP may evoke a dual response, i.e. when acting on P2X ligand-gated cation channels on smooth muscle cells it evokes transient depolarization, and thus an excitatory junction potential (EJP) (Stjarne, 1986), and when acting on endothelial G-protein-coupled P2Y receptors, it evokes hyperpolarizations (Keef et al. 1992).
Endothelial cells release an as yet unidentified endothelium-derived hyperpolarizing factor (EDHF) (Taylor & Weston, 1988; Garland et al. 1995; Malmsjö et al. 1998, 1999). This factor was also released by ATP in rabbit carotid (Chen & Suzuki, 1991), and guinea-pig and rabbit coronary (Keef et al. 1992) arteries. Thus, if the endogenously released ATP diffuses as far as the endothelium, it could enhance the release of EDHF. Since the neural plexus is situated in the adventitio-medial junction (Burnstock & Costa, 1975), if the diameter of the vessel is small with minimum layers of smooth muscle cells, the neural-endothelial separation narrows. Arteries about 150 µm in diameter consist of, on average, 1·5 smooth muscle cell layers (Neild, 1983), and the main endothelial vasoactive agent in small arteries is EDHF (Hwa et al. 1994). In such an artery, the transmitter (ATP) released from nerves could diffuse and act directly on the endothelial cells. The possible presence of such an interaction is also emphasized by Ralevic & Burnstock (1996a). However, to our knowledge no studies have been carried out to investigate the possibilities of endogenously released ATP exerting an effect on endothelial cells.
The aim of the present study was to determine whether or not the perivascular neurotransmitter in hamster mesenteric artery is ATP, and to investigate whether or not the ATP released from perivascular nerve stimulates the endothelium to release EDHF. The electrical events elicited by nerve stimulation were recorded using microelectrode techniques in mesenteric thin (100-150 µm) and thick (300-350 µm) arteries, and the differences were compared. In addition, electrical events elicited by exogenous ATP were studied. We show for the first time that neurally released ATP stimulates the endothelium to release EDHF. A preliminary account of these data has been presented previously (Thapaliya et al. 1999).
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METHODS |
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Tissue preparation
Male Golden Syrian hamsters (8-10 weeks old) were killed by an overdose of diethyl ether and exsanguinated, following a protocol approved by the Gifu University, Animal Care and Use Committee in accordance with Japanese Department of Agriculture guidelines. The superior mesenteric artery was removed with its branches of the iliac region. Its vascular bed was then separated from the intestine by cutting close to the intestinal wall, and placed in a Petri dish filled with physiological salt solution (PSS) at room temperature. The artery was cannulated and gently flushed with 1 ml of PSS in order to eliminate blood in the vessel. In some arterial segments, care was taken to preserve the endothelium. When required, the endothelium was removed by perfusion of warmed PSS containing collagenase (1 mg ml-1) for 15 min into the lumen of the vessels. Preparations were allowed to equilibrate for at least 1 h following removal of the endothelium (Kotecha & Neild, 1988). The removal of endothelial cells was considered to be successful when no hyperpolarization was elicited by acetylcholine. To observe changes in the membrane potential, arteries of about 100-350 µm (outside diameter) were used. For convenience, 300-350 µm diameter arteries are hereafter referred to as 'thick arteries' and the finer arterial branches of 100-150 µm diameter entering the intestine as 'thin arteries'.
Electrophysiological experiments
Intracellular recordings of membrane potential in arteries were made using glass filament microelectrodes filled with 3 M KCl with tip resistances ranging from 50 to 100 M
. An experimental partition chamber (capacity 3 ml) had a rubber base attached to the bottom, and was superfused at a rate of 3 ml min-1. The arteries were placed in the partition chamber in which large extracellular silver-silver chloride plates were used to elicit nerve stimulation, as described previously (Bolton et al. 1984). In all experiments, nerves were stimulated with a square-wave pulse (1 ms in duration) delivered at supramaximal intensity by a stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan). Repetitive nerve stimulation was applied using frequencies ranging from 1 to 20 Hz with a train of 6-100 pulses. Impalements were made from the adventitial side, within 2 mm from the stimulation electrode. In some experiments, when ATP was applied to the adventitial surface, the artery was tied at both ends by a thread to prevent the direct luminal contact of the drug. To perform the impalements from the intimal side, the artery was cut open transversely with fine iredectomy scissors and pinned adventitial side down in the experimental chamber. The electrical activities were monitored on an oscilloscope (CS 4026, Kenwood, Tokyo, Japan) and recorded on a thermal-array recorder (RTA-1100 M, Nihon Kohden) and on a PCM data recorder (RD-111T, TEAC, Tokyo, Japan) to allow replay for further analysis.
Drugs and solutions
The composition of the physiological salt solution was (mM): Na+, 137; K+, 5·9; Ca2+, 2·5; Mg2+, 1·2; Cl-, 134; HCO3-, 15·4; H2PO4-, 1·2; glucose, 11·4. The solution in the supply reservoir was gassed continuously with a 95 % O2: 5 % CO2 gas mixture creating a pH of 7·2 and was warmed to 33-35° C.
The drugs used were as follows: adenosine 5'-triphosphate (ATP) disodium salt, 
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-methylene ATP (,-MeATP) lithium salt, 2-methylthio ATP (2-MeSATP), 8-phenyltheophylline (8-PT), collagenase, tetrodotoxin, acetylcholine chloride, noradrenaline (NA) hydrochloride, guanethidine sulphate and N 
-nitro-L-arginine methyl ester (L-NAME) (Sigma); atropine sulphate monohydrate, suramin sodium, prazosin hydrochloride and uridine-5'-triphosphate (UTP) trisodium salt (Wako Pure Chemicals Industries, Osaka, Japan); 1-amino-4-[ [4-[ [4-chloro-6-[ [3 (or 4)-sulphophenyl]-amino-1,3,5-triazin-2-yl] amino]-3-sulphophenyl] amino-9,10-dihydro-9,10-dioxo-2-anthracenesulphonic acid (cibacron blue F3GA) (Funakoshi, Tokyo, Japan); pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), pinacidil and indomethacin (RBI); iloprost (Schering, Aktiengesellschaft, Germany).
Pinacidil (10 mM) was dissolved in dimethylsulphoxide (DMSO; 100 %). Indomethacin (10 mM) was dissolved in an equimolar concentration of Na2CO3. Prazosin (10 mM) was dissolved in methanol (100 %). 8-PT (10 mM) was dissolved in 80 % methanol (v/v) containing 0·2 M NaOH at a concentration of 10 mM. All the other drugs were dissolved in distilled water. The drugs were serially diluted in the PSS solution to the required final concentrations just before the experiments. Drugs were applied by addition to the superfused PSS in the required concentrations.
Preparation of nitric oxide solution
A stock solution of NO was prepared by a modification of the method of Stark et al. (1991). Briefly, NO gas was injected into PSS which was previously deoxygenated by gassing with He for 2 h, to give stock solutions of NO ranging from 0·01 to 1·0 % (v/v).
Statistics
Data are shown as means ± S.E.M.; n indicates the number of separate arteries, and only one artery was used from one animal. Statistical analysis was performed with Student's unpaired t test, and a P value of < 0·05 was regarded as significant.
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RESULTS |
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The resting membrane potentials of the smooth muscle cells of hamster thin and thick mesenteric arteries were -64·5 ± 0·5 mV (n = 105) and -63·9 ± 1·4 mV (n = 70), respectively. When unstimulated, the smooth muscle cells in both tissues were electrically quiescent. Perivascular nerve stimulation evoked EJPs in smooth muscle cells in both tissues, which were abolished by tetrodotoxin (0·3 µM, n = 3, data not shown). The amplitudes of the EJPs were graded depending on the stimulus strength; with a single stimulus the EJP amplitude was 4·6 ± 0·6 mV (n = 15) in thin arteries. The EJPs had a latency (time between stimulation and reaching 10 % of maximum amplitude) of 22·1 ± 1·0 ms, reached a peak amplitude 44·5 ± 2·4 ms after the stimulus, and had a total duration of 683·3 ± 33·3 ms. These values were similar to those recorded from thick arteries (amplitude 4·1 ± 1·0 mV, latency 22·4 ± 0·9 ms, reaching a maximum amplitude of 44·7 ± 2·5 ms after the stimulus, with a total duration of 684·1 ± 34·0 ms, n = 10).
Effects of suramin, PPADS and ,-MeATP on EJPs evoked by trains of brief stimuli
The mean amplitude of the fifth EJP evoked with a train of pulses at 1 Hz was 7·5 ± 0·2 mV (n = 12) in thin arteries. Suramin (100-700 µM), a P2 receptor antagonist, caused concentration-dependent inhibition of EJPs (Fig. 1A) without changing the resting membrane potential (-65·2 ± 1·1 mV, n = 4, P > 0·05). PPADS (0·5-5 µM), a P2X receptor antagonist, also inhibited the EJPs in a concentration-dependent manner (Fig. 1B), without affecting the resting membrane potential (-64·7 ± 0·4 mV, n = 10, P > 0·05). ,-MeATP (1 µM), a stable analogue of ATP, evoked a peak membrane depolarization of 21·7 ± 1·3 mV (n = 10). No other antagonistic drugs changed the membrane potential at the concentrations used in the present experiments. ,-MeATP (1 µM) strongly inhibited the EJPs after 60 min of treatment (control 7·1 ± 0·2 mV, n = 15, and ,-MeATP 0·9 ± 0·1 mV, n = 10, P < 0·001). The effects of suramin (700 µM), PPADS (5 µM) and ,-MeATP (1 µM) on EJPs of thick arteries were similar to those observed in thin arteries (control 7·3 ± 0·5 mV, n = 15, and suramin 0·9 ± 0·1 mV, n = 4; control 7·1 ± 0·5 mV, n = 6, and PPADS 0·4 ± 0·1 mV; control 6·9 ± 0·4 mV, n = 12, and ,-MeATP 0·5 ± 0·2 mV, n = 5).
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EJPs evoked by nerve stimulation at a frequency of 1 Hz (Aa and Ba control) were abolished by suramin (700 µM) treated for 90 min (Ab) and PPADS (5 µM) treated for 25 min (Bb). The vertical lines represent the stimulation artifact. Stimulus was a supramaximal voltage, 1 ms in duration. The responses shown in A and B were recorded from two different cells and membrane potentials were -63 and -65 mV, respectively. Ac and Bc are summaries of the effects of suramin and PPADS, respectively, on EJP amplitude in thin arteries. Each column and bar represents mean ± S.E.M. (n = 9-12). * Significantly different from control (P < 0·001). | ||
Effect of prazosin on slow depolarization evoked by trains of nerve stimulation
In thick arterial preparations, when a train of pulses at 1 Hz was applied, each pulse evoked an EJP. As the train progressed, after the third or fourth pulse, a slow sustained depolarization (2·5 ± 0·9 mV, in 5/25 preparations) appeared which continued after the cessation of stimulation, then slowly declined over 30-40 s (Fig. 2Aa). Such trains of low-frequency (
1 Hz) stimuli failed to evoke slow depolarization in thin arteries. In all the thick and 49/63 thin arterial preparations, trains of high-frequency (20 Hz) stimuli evoked biphasic depolarizations consisting of summed EJPs of 19·3 ± 0·9 mV (n = 12), followed by slow depolarizations of 5·2 ± 0·2 mV (n = 11), which gradually returned to resting potential level after 41·2 ± 1·4 s (n = 11) from the end of the stimulation (Fig. 2B a). The peak of slow depolarization was observed about 2 s after the cessation of stimulation. Prazosin (1 µM), a selective 1-adrenoceptor antagonist, abolished the slow depolarization evoked by trains of pulses (at all frequencies), but did not affect the amplitude of EJPs evoked by either 1 Hz or 20 Hz stimulation (n = 6, Fig. 2Ab and Bb). Methanol (0·01 %) at the concentration used to prepare prazosin (1 µM) had no effect on either membrane potential or slow depolarization (n = 4, data not shown).
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Nerve stimulation of 6 pulses at 1 Hz in thick artery (A) and 50 pulses at 20 Hz in thin artery (B) evoked EJPs followed by slow depolarization. The slow depolarizations in Aa and Ba (control) were abolished by prazosin (1 µM) applied for 30 min (Ab and Bb). The responses shown in Aa and Ab were recorded from the same cell, but Ba and Bb were recorded from different cells. The vertical lines represent the stimulation artifact. Stimulus was a supramaximal voltage, 1 ms in duration. Membrane potentials for Aa and Ba were -61 and -64 mV, respectively. The dashed lines mark the resting membrane potential. | ||
Properties of hyperpolarization evoked by trains of nerve stimuli
In 14/63 thin arterial preparations, nerve stimuli with a train of pulses at 20 Hz evoked a biphasic change in membrane potential consisting of summed EJPs, followed by a transient hyperpolarization which occurred when the stimulation ceased. With 25-100 pulses at 20 Hz, the amplitude of transient hyperpolarization reached a maximum level at a train of 50 pulses and so we used this stimulus pattern for the rest of the experiments. In this series of experiments, prazosin (1 µM) was used to prevent slow depolarization. Guanethidine (5 µM, n = 4, Fig. 3Ab) and tetrodotoxin (0·3 µM, n = 4, data not shown) abolished depolarizations and hyperpolarizations induced by nerve stimulation. PPADS (5 µM), a P2X receptor antagonist, strongly inhibited the summed EJPs from 19·5 ± 1·1 mV (n = 15) before, to 0·7 ± 0·3 mV (n = 12, P < 0·001) after, drug treatment. In PPADS-treated thin arterial preparations, transient hyperpolarizations (4·5 ± 0·9 mV, n = 20, Fig. 3B a) were evoked by nerve stimulation. However, in the thick artery the hyperpolarization was never evoked even with intimal impalement (n = 5, data not shown). The remaining description will be restricted to the responses of the thin arteries treated with PPADS (5 µM), prazosin (1 µM) and atropine (1 µM).
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A was recorded in the presence of prazosin (1 µM). In Aa summed EJPs were followed by the transient hyperpolarization evoked by trains of stimuli (50 pulses at 20 Hz). Guanethidine (5 µM) inhibited both summed EJPs and transient hyperpolarization (Ab). B was recorded in the presence of PPADS (4 µM), prazosin (1 µM) and atropine (1 µM). The transient hyperpolarization evoked by a train of stimuli (50 pulses at 20 Hz) (Ba) was abolished by cibacron blue (CB) F3GA (100 µM) (Bb). Stimulus was a supramaximal voltage, 1 ms in duration. Membrane potentials for A and B were -65 and -67 mV, respectively. Bc is a summary of the effect of CB F3GA (2-100 µM) on nerve stimulation-evoked transient hyperpolarization. Each column and bar represents mean ± S.E.M. (n = 7-10). * Significantly different from control (P < 0·001). | ||
Effect of cibacron blue F3GA on the hyperpolarization evoked by nerve stimuli
The type of receptor mediating the release of hyperpolarizing factor in response to nerve stimulation was assessed by employing cibacron blue F3GA (2-100 µM), a P2Y receptor antagonist. It inhibited the transient hyperpolarizations evoked by trains of nerve stimuli in a concentration-dependent manner (Fig. 3B c). Nucleotidase, which is present in smooth muscle tissue, can produce adenosine from ATP. Therefore, the possible involvement of adenosine in nerve stimulation-induced hyperpolarization was investigated using 8-PT, an adenosine receptor antagonist. 8-PT did not affect the hyperpolarization evoked by nerve stimulation (4·7 ± 0·4 mV, n = 6, P > 0·05). These results indicated that the transient hyperpolarizations were evoked by the activation of P2Y receptors. Pinacidil (0·2 µM)-induced hyperpolarization was not significantly changed by cibacron blue F3GA (100 µM) (control 12·4 ± 1·1 mV, n = 6, and cibacron blue F3GA 10·0 ± 1·5 mV, n = 6, P > 0·05). DMSO (0·05 %), at the concentration used to prepare pinacidil (0·2 µM), had no effect on either membrane potential or hyperpolarization (n = 4, data not shown).
Effect of endothelium denudation on the hyperpolarization evoked by trains of nerve stimuli
The involvement of the endothelium in the nerve stimulation-induced hyperpolarization was studied. Endothelium-denuded arteries were electrically quiescent when not stimulated, but the resting membrane potential was significantly depolarized from -64·5 ± 0·5 (n = 105) in endothelium intact arteries to -61·5 ± 1·0 mV (n = 40, P < 0·01). The nerve stimulation-induced hyperpolarization was not observed after endothelium denudation (before 5·2 ± 0·7 mV, n = 5, and after 0·9 ± 0·5 mV, n = 5, P < 0·001; Fig. 4Ab), but the amplitudes of EJPs were unaltered by endothelium denudation in a separate series of experiments (n = 5, not significantly different, data not shown). Although it has been reported that the denudation of endothelial cells in such a narrow diameter artery damages the smooth muscle, the responses of smooth muscle to ,-MeATP or NA were unaltered by endothelium denudation. Similarly, hyperpolarizing responses to nitric oxide (1 µM, 7·8 ± 1·2 mV, n = 4) and iloprost (1 µM, 7·3 ± 1·8 mV, n = 9) were not affected by the endothelial denudation (7·2 ± 0·9 mV, n = 3, P > 0·05 and 7·0 ± 1·4 mV, n = 4, P > 0·05, respectively). Therefore, smooth muscle cell function was intact after denudation of the endothelium.
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The response shown in A was recorded in the presence of PPADS (4 µM), prazosin (1 µM) and atropine (1 µM). A transient hyperpolarization was evoked by a train of stimuli (50 pulses at 20 Hz) in Aa, which was not observed after endothelium denudation in Ab. The responses shown in B and Ca were recorded in the presence of | ||
Effects of L-NAME and indomethacin on the hyperpolarization evoked by trains of nerve stimuli
Involvement of NO and prostanoid in the nerve stimulation-induced hyperpolarization was investigated. L-NAME (100 µM), an NO synthase inhibitor, and indomethacin (5 µM), a cycloxygenase inhibitor, together did not change the resting membrane potential (-63·9 ± 0·9 mV, n = 12, P > 0·05) or nerve stimulation-evoked hyperpolarization (4·3 ± 0·4 mV, n = 12, P > 0·05), suggesting no involvement of NO or prostanoid. Na2CO3 (5 µM), at the concentration used to prepare indomethacin (5 µM) had no effect on either membrane potential or the nerve stimulation-induced hyperpolarization (n = 3, data not shown).
Adventitial application of ATP in ,-MeATP-desensitized thin and thick arteries
Adventitial application of ATP (100 µM) for 1 min evoked a hyperpolarization of 7·8 ± 1·1 mV (n = 6, Fig. 4B a), which occurred after a delay of 10-15 s in ,-MeATP-desensitized thin artery. Time to peak ranged between 40 and 50 s and membrane potential returned to resting level after 1 min of washout. In contrast, a membrane potential response was not evoked in thick artery (n = 7, Fig. 4C a) with adventitial surface application of ATP (100 µM). The hyperpolarization of thin artery was not observed when the endothelium was denuded (n = 6, Fig. 4B b).
Intimal application of P2 receptor agonists
The type of receptor involved in purinoceptor agonist-evoked hyperpolarization was investigated. Impalements were performed from the intimal side and the results are summarized in Table 1. Intimal application of ATP (100 µM) for 1 min evoked a hyperpolarization after 2-5 s of application. Time to peak ranged between 30 and 40 s and returned to resting level after 1 min of washout. In the presence of cibacron blue F3GA (100 µM) or after endothelial denudation, the ATP-evoked hyperpolarization was converted to a depolarization. Intimal application of UTP (100 µM), a P2Y2-like receptor (previously P2U receptor) agonist, evoked a hyperpolarization. This hyperpolarization was abolished when the endothelium was denuded, and significantly inhibited when the vessels were treated with cibacron blue F3GA for 30 min. L-NAME and indomethacin together did not modify the UTP-evoked hyperpolarization. Similarly, PPADS (5 µM) did not change the UTP-evoked hyperpolarization. On the other hand, application of 2-MeSATP (7 µM) evoked a depolarization. There was no effect of endothelium denudation on the 2-MeSATP-evoked depolarization.
Table 1. Effects of various agents and endothelium denudation on membrane potential response (± mV) with intimal application of P2 receptor agonists
| ATP (100 µM) | UTP (100 µM) | 2-MeSATP (7 µM) | |
| Control (with endothelium) | -12·8 ± 0·6 (n = 5) | -11·4 ± 1·0 (n = 5) | 6·4 ± 0·2 (n = 5) |
| Without endothelium | 12·7 ± 1·4 (n = 4) * | 0 (n = 4) * | 7·1 ± 1·0 (n = 3) |
| Cibacron blue (100 µM) | 12·4 ± 3·3 (n = 5) * | -2·5 ± 0·5 (n = 4)  |
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| PPADS (5 µM) | - | -11·7 ± 0·9 (n = 5) | - |
| Indomethacin (5 µM) + L-NAME (100 µM) |
- | -12·1 ± 0·3 (n = 4) | - |
P < 0·05.
Overall, the results shown in Table 1 indicate that P2Y2-like receptors located on endothelium are involved in the ATP- and UTP-evoked hyperpolarization.
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DISCUSSION |
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The present experiments provide evidence that ATP released from nerve terminals innervating hamster mesenteric arteries induces the release of EDHF from endothelial cells. Neurally released ATP evoked biphasic responses that were initially EJPs (a direct action) followed by EDHF-induced hyperpolarization (an indirect action) on the smooth muscle. This is the first demonstration of neurally released ATP mediating hyperpolarization through EDHF. This conclusion is based on the results obtained from the present experiments, the most substantial of which was the abolition of hyperpolarization by endothelial denudation and inhibition by an endothelial P2Y receptor antagonist.
Suramin, a non-selective antagonist of P2 receptors (Dunn & Blakeley, 1988; Hoyle et al. 1990) inhibits purinergic EJPs (Sneddon, 1992; McLaren et al. 1995). It was also reported that PPADS is a specific P2X receptor antagonist in rat tail isolated artery (McLaren et al. 1998). Similarly, it has also been reported that the desensitization of P2 receptors by ,-MeATP reduces or abolishes electrical and contractile responses evoked by purinergic nerve stimulation in various vascular tissues (Ishikawa, 1985; Sneddon & Burnstock, 1985; Miyahara & Suzuki, 1987; Ramme et al. 1987). In the present experiments, EJPs recorded from hamster mesenteric arteries were inhibited by suramin, PPADS or ,-MeATP. These findings suggest that EJPs in hamster mesenteric arteries are mediated by ATP or purine nucleotide. In other blood vessels, it is suggested that ATP is co-released with NA (Sneddon & Burnstock, 1985; Kugelgen & Starke, 1985). Slow -adrenoceptor-mediated depolarization following EJPs in rat tail arteries (Sneddon & Burnstock, 1985) is reported. In hamster mesenteric arteries, trains of stimuli evoked prazosin-sensitive slow depolarizations with slower time courses and smaller amplitudes than those of the summed purinergic responses. It has been reported that at higher frequencies the vasoconstrictor response to NA is enhanced (Burnstock & Warland, 1987a; Schwartz & Malik, 1989). In the present experiments, low-frequency trains of stimuli evoked slow depolarization in 20 % of thick arteries, but the high-frequency trains of stimuli consistently evoked prazosin-sensitive slow depolarizations in both kinds of arteries. We interpreted this to mean that the adrenergic innervation in thick arteries is denser than in thin arteries.
Inhibitory neurotransmission has been reported in different blood vessels (Neild et al. 1990; Toda & Okamura, 1992; Kotecha & Neild, 1995). It has also been reported that cholinergic nerve stimulation induced hyperpolarization or vasodilatation in arterioles of guinea-pig submucosal plexus, choroidal arterioles and rabbit lingual artery (Brayden & Large, 1986; Neild et al. 1990; Hashitani et al. 1998). As we have conducted the experiments in the presence of atropine, we can exclude the possibility of involvement of muscarinic cholinoceptors. It was also reported that in many cerebral arteries, atropine-insensitive neurogenic vasodilatation appears to be due to an NO-related compound released from non-adrenergic, non-cholinergic (NANC) nerve endings (Toda & Okamura, 1990; Toda et al. 1990). If NO was derived from NANC nerves, NO-mediated hyperpolarization would be unaffected by endothelial denudation of the vessels, because the exogenous NO hyperpolarized the smooth muscle cells in both endothelium- intact and -denuded preparations of thin arteries. However, the perivascular nerve stimulation-induced hyperpolarization was observed in neither endothelium-denuded thin artery nor intact thick artery. Nerve stimulation-evoked hyperpolarization still persisted in L-NAME-treated preparations. Thus, in the present experiments, NO from nitrergic nerves seems unlikely to be involved.
In hamster mesenteric artery, the transient hyperpolarization was abolished by tetrodotoxin, indicating that it originated from neurones. However, the release of a hyperpolarizing and relaxing substance from endothelial cells directly in response to field stimulation is reported in monkey coronary artery (Mekata, 1986) and bovine pulmonary artery (Buga & Ignarro, 1992), respectively. If such a substance is released from endothelial cells, the response should be recorded in both types of arteries. However, in the present experiments, we were unable to obtain hyperpolarizing responses in thick artery even with intimal impalement.
In the present experiments, the nerve stimulation-induced hyperpolarization was inhibited by blocking sympathetic nerve activity with guanethidine and also by antagonizing the purinoceptors with cibacron blue F3GA. These results indicate that the response may be evoked by activation of purinergic neurones and P2Y receptors. The significant inhibition of the transient hyperpolarization by the purinoceptor antagonist is unlikely to be due to a non-specific action, because this antagonist has been demonstrated to be a selective P2Y receptor antagonist in vascular tissues (Burnstock & Warland, 1987b; Hopwood & Burnstock, 1987). Furthermore, cibacron blue F3GA did not modify pinacidil-induced hyperpolarization. P2Y receptors are typically located on the endothelium (O'Connor et al. 1991). The nerve stimulation-induced hyperpolarization observed in thin arteries was mimicked by exogenous ATP. Both P2 receptor agonist- and nerve stimulation-induced hyperpolarizations were abolished by endothelium denudation. Therefore, it could be concluded that the neurally released ATP evokes endothelium-mediated hyperpolarization. ATP-stimulated NO release occurred in endothelial cells from the guinea-pig pulmonary artery (Liu et al. 1992). Stimulation of endothelium with acetylcholine is reported to release prostacyclin in guinea-pig coronary artery (Parkington et al. 1993). But in our experiments, treatment with L-NAME and indomethacin did not modify the nerve stimulation-induced hyperpolarization suggesting that the involvement of NO and prostacyclin is unlikely. In guinea-pig and rabbit coronary arteries, Keef et al. (1992) have reported that ATP-induced hyperpolarization was mediated by EDHF, which is consistent with our results. Therefore, it is suggested that ATP released from nerve terminals diffuses into the endothelium where it activates P2Y receptors to induce the release of EDHF in thin arteries.
One can argue that the activation of P1 receptors located on smooth muscle can produce hyperpolarization by adenosine, which is the metabolite of ATP, because endothelial cells possess high nucleotidase activity, and that the enzyme could produce AMP or adenosine from ATP during superfusion. However, the production of such metabolites needs several minutes after the start of the perfusion of ATP (Gorden, 1986). In the present experiments, the nerve stimulation-induced hyperpolarization was unlikely to be due to a metabolite of ATP, since the responses occurred within a few seconds. Moreover, 8-PT, a potent P1 purinoceptor antagonist (Griffith et al. 1981) had no effect on the hyperpolarization.
Nerve stimulation induced hyperpolarization in arteries < 150 µm diameter but not in those > 300 µm diameter, while the amplitudes of the EJPs and slow depolarizations were similar in both kinds of arteries. In addition, we were unable to record hyperpolarization in response to adventitial application of ATP in thick artery. Presumably, this latter result suggests that a sufficient concentration of ATP did not reach the endothelium to evoke hyperpolarization. The number of smooth muscle cell layers within the media of small arteries decreases in parallel with the decrease in vessel diameter from approximately six layers in 300 µm vessels (Lee et al. 1982) to an average of 1·5 layers in 150 µm vessels (Neild, 1983).
The potency for the hyperpolarizing effects of purinergic agonists in hamster mesenteric arteries is ATP = UTP. This order is compatible with the results of a previous study (Ralevic & Burnstock, 1996b), and the equipotency of ATP and UTP as vasodilators is indicative of actions at the P2Y2-like receptor (previously P2U receptors) (O'Connor et al. 1991; Ralevic & Burnstock, 1998). Although 2-MeSATP is taken as a potent and selective agonist at the P2Y1-like receptor (Ralevic & Burnstock, 1998), it also acts on P2X1-like receptors in single rat tail artery smooth muscle cells (Evans & Kennedy, 1994). In the present experiments, intimal application of 2-MeSATP did not cause hyperpolarization, suggesting that P2Y1-like receptors play a minimal role in hyperpolarization induced by a purinergic agonist. The depolarization induced by this agent may be due to activation of the P2X1-like receptor. Taken together, these results indicate that hamster mesenteric arterial endothelium contains P2Y2-like receptors, and that these can be antagonized by cibacron blue F3GA, which significantly inhibited UTP-induced hyperpolarization.
In conclusion, the present findings demonstrate that ATP released from the perivascular nerves may diffuse as far as the endothelium and activate P2Y2-like receptors to induce the release of EDHF in thin arteries, but not in thick arteries. EDHF appears to be a primary functional factor in small arteries, thus we propose that EDHF released by neural ATP may play an important physiological role in the local regulation of vascular resistance.
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| Corresponding author | |
| T. Takewaki: Department of Pathogenetic Veterinary Science, The United Graduate School, Gifu University, Gifu 501-1193, Japan. |
