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NEUROSCIENCE |
1 Laboratory of Pharmacology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
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Abstract |
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(Received 13 March 2006;
accepted after revision 28 May 2006;
first published online 1 June 2006)
Corresponding author K. Otsuguro: Laboratory of Pharmacology, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Japan. Email: otsuguro{at}vetmed.hokudai.ac.jp
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Introduction |
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Adenosine mediates several functions via the activation of A1, A2A, A2B and A3 adenosine receptor subtypes and mainly inhibits neurotransmission via A1 receptors in the brain (Fredholm et al. 2001). In the spinal cord, adenosine also has an inhibitory effect on synaptic transmission in vitro (Li & Perl, 1994; Nakamura et al. 1997; Lao et al. 2004), and thus mediates antinociception in vivo (Sawynok, 1998; Sawynok & Liu, 2003). In nociceptive tests of mice and rats, it is also shown that analgesia is produced by the intrathecal administration of adenosine receptor agonists or metabolic inhibitors of adenosine such as adenosine kinase inhibitors (Post, 1984; Poon & Sawynok, 1995; Kei & DeLander, 1996).
Acidosis is reported to have inhibitory effects on neuronal activity in the brain. It is reported that low extracellular pH (pHo) inhibits voltage-dependent Na+ and Ca2+ channels (Tombaugh & Somjen, 1996) and glutamate NMDA receptors (MacBain & Mayer, 1994). Therefore, it is generally accepted that the inhibition of these channels and receptors results in the suppression of synaptic transmission in the CNS. Although an acute increase in carbon dioxide (CO2), i.e. hypercapnia, immediately evokes synaptic depression in rat hippocampal slices (Balestrino & Somjen, 1988; Lee et al. 1996; Velí
ek, 1998; Hsu et al. 2000), it leads to a rapid fall not only in pHo but also in the intracellular pH (pHi) of brain tissue (Martoft et al. 2003). Because hypercapnia induces antinociception, it is used to promote preslaughter anaesthesia in livestock and short-lasting anaesthesia in, or killing of, laboratory animals (Mischler et al. 1996; Martoft et al. 2002).
Ischaemia/hypoxia is well known to release adenosine, which cause vasodilatation and depression of neuronal excitability in the CNS (Wardas, 2002; O'Regan, 2005). On the other hand, it has been reported that hypercapnic acidosis releases adenosine and evokes vasodilatation via the activation of A2A receptors in rat coronary (Phillis et al. 1998) and cerebral vessels (Phillis et al. 2004). Recently, Dulla et al. (2005) have also reported that hypercapnic acidosis releases adenosine and depresses neuronal activity in a rat hippocampal slice preparation. If this is the case in the spinal cord, hypercapnic acidosis may release adenosine, resulting in the depression of spinal synaptic transmission and thus antinociception. However, it is unclear whether adenosine contributes to the effect of hypercapnic acidosis on the spinal neuronal activities.
To investigate the effect of hypercapnic acidosis on spinal transmission, we exposed an isolated spinal cord preparation to artificial cerebrospinal fluid gassed with 20% CO2. Under this condition, the pH in the solution decreased to 6.7, which may occur under pathological conditions such as ischaemia (Tombaugh & Sapolsky, 1993) or respiratory acidosis (Martoft et al. 2003). We herein demonstrate that the activation of adenosine A1 receptors is involved in the hypercapnic acidosis-evoked depression of reflex potentials in the isolated spinal cord of the neonatal rat. We suggest that a fall in pHi inhibits adenosine kinase activity and then causes the accumulation of extracellular adenosine.
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Methods |
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All experiments were approved by the Animal Research Committee of the Graduate School of Veterinary Medicine, Hokkaido University. All efforts were made to minimize animal suffering and to reduce the number of animals used. Both male and female neonatal rats (Wistar, 0–3 days old) were used in this experiment. Neonatal rats were anaesthetized with diethyl ether and decapitated, and then the spinal cords were isolated.
Electrophysiology
The isolated spinal cord preparation was prepared as previously described (Otsuguro et al. 2006). The hemisected spinal cord was superfused with artificial cerebrospinal fluid (ACSF) at a flow rate of about 2.5 ml min–1 at 27 ± 2°C. Suction stimulating and recording electrodes were placed on the dorsal and ipsilateral ventral roots (L3–L5), respectively. Electrical stimulation of the dorsal root elicited a monosynaptic reflex potential (MSR) followed by a slow ventral root potential (sVRP) lasting about 20 s. The magnitude of MSR was expressed as the peak amplitude (mV), and sVRP was expressed as the integral of depolarization (mV s) over the resting potential. The dorsal root was stimulated every 2 min by a single square wave pulse of 0.3–0.7 ms duration and 30–40 V amplitude (a supramaximal intensity for sVRP) throughout the experiments. The preparation was allowed to equilibrate for about 1 h before recordings. Compound action potential (CAP) was recorded from the central cut end of the L3 dorsal root. The L3 dorsal root was cut near the spinal cord and the L3 lumbar nerve was isolated together with the dorsal root. When the lumbar root was electrically stimulated (50 V, 0.3–0.8 ms), CAP measured from the L3 dorsal root consisted of two waves, which were probably mediated by A and C primary afferent fibres. The magnitude of CAP was expressed as the peak amplitude (mV). The effects of acidosis/hypercapnia and drugs on spinal reflex potentials and CAP were evaluated by the mean of three responses that had become stable after treatment, and expressed as a percentage of the mean of two control responses just before treatment. The time course of the magnitude of these responses was expressed as a percentage of the control response. Electrical responses were detected with high gain amplifiers (MEZ-8300 and 8301, Nihon Kohden, Tokyo, Japan). MSR and CAP were recorded using a thermal arraycorder (WR7900, Graftec, Yokohama, Japan) with a sampling time of 80 µs. The sVRP was digitized by an analog/digital converter (PowerLab, ADInstruments, Castle Hill, Australia) with a sampling time of 10 ms. Data were stored in a personal computer and analysed thereafter with software (Chart 5, ADInstruments).
Measurement of adenosine concentration
The spinal cord isolated from the neonatal rat was cut into five pieces and preincubated for 1 h at 35°C. Then all pieces of the spinal cord were incubated in a tube containing normal ACSF (1 ml) for 10 min at 35°C and 250 µl of ACSF was collected as a control. After the external solution was changed to hypercapnia/acidosis solution (1 ml), the tissue was incubated for another 10 min. Then an aliquot (250 µl) of the solution was taken as a treated sample and the pieces of tissue were weighed after blotting. The average wet weight of the isolated spinal cord was 28.1 ± 2.1 mg (n
= 17). The adenosine concentration in the samples was determined by high-performance liquid chromatography (HPLC) according to the method of Kawamoto et al. (1998) with some modifications. The collected samples (250 µl) were immediately chilled on ice, and 90 µl of 0.1 M citrate–phosphate buffer (pH 4.0) and 10 µl of 40% chloroacetaldehyde were added to the sample. A 25 µl solution of 10 µM
,ß-methylene ADP (AOPCP) was also added as an internal standard. The samples were then incubated at 80°C for 40 min to make ethenoadenosine derivatives, which were measured by reverse-phase HPLC with a Cosmosil 5C18-MS column (4.6 mm x 150 mm, Nacalai Tesque Inc., Kyoto, Japan) equipped with a fluorescence detector (FP-540D, Nihon-Koden, Tokyo, Japan). The wavelengths for excitation and emission were set at 270 and 420 nm, respectively. The mobile phase buffer consisted of 100 mM KH2PO4, 5 mM tetrabutylammonium bromide and 2.0% CH3CN adjusted to pH 3.3 with H3PO4. The amount of adenosine in a sample was quantified by direct comparison of the peak height to that of the internal standard.
Measurement of adenosine kinase activity
Adenosine kinase activity of the isolated spinal cord was assessed by measuring the in vivo phosphorylation of [U-14C]adenosine according to methods of Ives et al. (1969) and Lynch et al. (1998) with some modifications. The isolated spinal cord was cut into 3–4 pieces and preincubated with normal ACSF for 1 h at 35°C. Then each piece was separately incubated with normal ACSF or hypercapnia/acidosis solution (1 ml) containing 0.2 µM [U-14C]adenosine (17.3 GBq mmol–1, Amersham Biosciences, Piscataway, NJ, USA) for 10 min at 35°C. Then samples were boiled to terminate the reaction. After chilling, samples were homogenized and centrifuged. The supernatant (100 µl) was spotted onto ion exchange paper disks (Whatman DE-81, Whatman, Brentfold, UK). The disks were allowed to stand for 10 min and then washed with 2 mM ammonium formate, rinsed successively with distilled water, methanol and acetone, dried in room air, and exposed to a solution of 0.1 M HCl–0.4 M KCl. The radioactivity of nucleotides bound to the paper was measured with a liquid scintillation counter. The content of protein was measured by the method of Bradford (1976). The average protein content of the spinal cord piece was 0.43 ± 0.02 mg (n = 40). The effects of hypercapnia/acidosis on adenosine kinase activity were expressed as the percentage inhibition relative to the control value obtained from another piece of the spinal cord of the same rat.
Solutions
The composition of ACSF was as follows (mM): NaCl 138, NaHCO3 21, NaH2PO4 0.6, CaCl2 1.25, KCl 3.5, MgCl2 2.0, glucose 10. The ACSF was equilibrated with a gas mixture of 95% O2 and 5% CO2, pH 
7.3. The composition of hypercapnic acidosis solution (20% CO2, pH 6.7) was the same as that of the ACSF solution with the exception that it was equilibrated with a gas mixture of 80% O2 and 20% CO2, pH 6.7. For the isocapnic acidosis solution (5% CO2, pH 6.7), NaHCO3 was decreased to 5 mM (replacing equimolar NaCl) with a gas mixture of 95% O2 and 5% CO2, pH 6.7. For the isohydric hypercapnia solution (20% CO2, pH 7.3), NaHCO3 was increased to 80 mM (replacing equimolar NaCl) with a gas mixture of 80% O2 and 20% CO2, pH 7.3.
Materials
5'-Amino-5'-deoxyadenosine p-toluenesulphonate salt (NH2dAD), 8-cyclopentlyl-1,3-dimethylxanthine (CPT) and ,ß-methylene adenosine 5'-diphosphate sodium salt (AOPCP) were purchased from Sigma (St Louis, MO, USA). 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385) and erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) were from Tocris (Bristol, UK). L-Glutamic acid hydrochloride was from Tokyo Kasei Kogyo (Tokyo, Japan).
Data analysis
Results are expressed as means ± S.E.M. Statistical comparisons between two groups were performed by Student's paired t test. A P value of less than 0.05 was considered significant.
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Results |
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We investigated the effect of hypercapnic acidosis (20% CO2, pH 6.7) on the spinal reflex potentials evoked by dorsal root stimulation at 2 min intervals. The baseline ventral root potential was slightly depolarized by hypercapnic acidosis for 10 min (Fig. 1A and Table 1). In addition, hypercapnic acidosis reversibly decreased the amplitude of MSR and the integral of sVRP (Fig. 1B), which reached the maximum 4–6 min after exposure to hypercapnic acidosis. MSR and sVRP reversed to the original level within 10 min after normal ACSF solution (Fig. 1C). The second and third exposures to hypercapnic acidosis solution with 10 min intervals resulted in reproducible depression of MSR to 46.1 ± 5.7% (n = 5, 1st), 46.7 ± 5.0% (n = 5, 2nd) and 40.7 ± 4.6% (n = 5, 3rd) and sVRP to 37.2 ± 8.9% (n = 5, 1st), 29.2 ± 7.5% (n = 5, 2nd) and 28.2 ± 8.4% (n = 5, 3rd).
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7.2), there was no effect on MSR (99.8 ± 0.4%, n
= 4) or sVRP (97.9 ± 5.6%, n
= 4). The spinal reflex potentials are mediated via the release of several neurotransmitters from presynaptic neurons (Nussbaumer et al. 1989; Woodley & Kendig, 1991), which is largely dependent on the external Ca2+. As shown in Fig. 2, the increase in the Ca2+ concentration from 1.25 to 2.5 mM in ACSF enhanced the magnitudes of MSR and sVRP. Under this condition, hypercapnic acidosis caused a small depression of the spinal reflex potentials. After returning the Ca2+ concentration to 1.25 mM, the magnitude of the spinal reflex potentials and the depression of hypercapnic acidosis almost recovered to the control level.
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To investigate whether hypercapnic acidosis affected the compound action potential (CAP), we examined the effect of hypercapnic acidosis on the dorsal root CAP evoked by electrical stimulation of the lumbar nerve (Fig. 3A and B). As shown in Fig. 3A, electrical stimulation evoked two waves with different conduction velocities. It has been reported that these originate from the excitation of A and C fibres (Faber et al. 1997). Exposure to the hypercapnic acidosis solution failed to influence the amplitude of either the 1st (100.1 ± 5.4%, n = 4) or 2nd wave (94.5 ± 6.4%, n = 4).
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Effects of adenosine receptor antagonists on depression of spinal reflex responses by hypercapnic acidosis
In the CNS, it is well known that ischaemia/hypoxia causes adenosine release, which depresses synaptic transmission through activation of adenosine receptors. Therefore, to examine whether adenosine was also involved in the hypercapnic acidosis-evoked depression of MSR and sVRP, we next investigated the effects of CPT and ZM241385, selective adenosine A1 and A2A receptor antagonists, respectively, on the hypercapnic acidosis-evoked depression (Fig. 4). Although the extent of depression by hypercapnic acidosis varied from preparation to preparation, it was reproducible in the same preparation. Therefore, the MSR and sVRP during the 1st exposure to hypercapnic acidosis were used as a control response. Then we pretreated a spinal cord with CPT (3 µM) or ZM241385 (0.3 µM) for 20 min and observed the MSR and sVRP during the 2nd exposure to hypercapnic acidosis in the presence of these drugs. The pretreatment with CPT did not cause changes in MSR (115.3 ± 7.3%, n = 4) or sVRP (97.5 ± 8.2%, n = 4). On the other hand, as shown in Fig. 4A, the hypercapnic acidosis-evoked depression of the spinal reflex responses was significantly reduced by CPT compared to the control. ZM241385 caused neither changes in MSR (104.7 ± 4.5%, n = 6) and sVRP (92.1 ± 4.0, n = 6) nor the hypercapnic acidosis-evoked depression of MSR and sVRP (Fig. 4B). The hypercapnic acidosis-evoked depolarization in the basal ventral root potential was not influenced by CPT or ZM241385 (Table 1).
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2 adrenoceptor antagonist) did not affect the hypercapnic acidosis-evoked depression of spinal reflex responses (data not shown). Effects of isocapnic acidosis and isohydric hypercapnia on spinal reflex responses
We also examined the effects of isocapnic acidosis (5% CO2, pH 6.7) and isohydric hypercapnia (20% CO2, pH 7.3) on MSR and sVRP. Although exposure to the isocapnic acidosis solution induced a slight depolarization of the basal ventral root potential to the same extent as hypercapnic acidosis (Table 1), it caused only weak depression of MSR and sVRP (Fig. 5A). On the other hand, isohydric hypercapnia markedly depressed MSR and sVRP, which were significantly reversed by CPT (Fig. 5B). In addition, the isohydric hypercapnia hyperpolarized a basal ventral root potential that was not significantly affected by CPT (Table 1).
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Ecto-5'-nucleotidase is an important enzyme for formation of extracellular adenosine via the breakdown of released adenine nucleotides. Therefore, to explore whether adenosine activating adenosine A1 receptors was due to their breakdown, we examined the effect of AOPCP, an inhibitor of ecto-5'-nucleotidase, on the hypercapnic acidosis-evoked depression of MSR and sVRP (Fig. 6). AOPCP (50 µM) increased spontaneous activities of the basal ventral root potential as shown in Fig. 5A, but did not affect MSR (105.1 ± 4.1%, n = 5) and sVRP (122.1 ± 6.9%, n = 5). In the presence of AOPCP (50 µM), the hypercapnic acidosis abolished the spontaneous activities. The hypercapnic acidosis-evoked depression of MSR and sVRP was not affected by AOPCP (50 µM). AOPCP at a higher concentration (200 µM) caused no effect on the hypercapnic acidosis-evoked depression of MSR (control: 53.7 ± 6.8%, n = 4, AOPCP: 49.2 ± 7.1%, n = 4) and sVRP (control: 27.1 ± 9.5%, n = 4, AOPCP: 34.4 ± 13.1%, n = 4).
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Adenosine kinase and deaminase are also important enzymes for regulating intracellular and extracellular adenosine concentrations. The inhibition of these enzymes is expected to result in accumulation of adenosine. Therefore, we investigated whether NH2dAD, an adenosine kinase inhibitor, and EHNA, an adenosine deaminase inhibitor, could mimic the inhibitory effect of hypercapnic acidosis. Like hypercapnic acidosis, NH2dAD (5–50 µM) depressed both MSR and sVRP in a concentration-dependent manner (Fig. 7A and C), and the NH2dAD-evoked depression of MSR and sVRP was reversed by CPT (3 µM). On the other hand, EHNA (10–100 µM) depressed sVRP but not MSR. In addition, CPT failed to recover the EHNA-evoked depression of sVRP (Fig. 7B and D).
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Exposure to the hypercapnic acidosis solution for 10 min caused the accumulation of extracellular adenosine (150.4 ± 16.6% of control, n = 6) in the spinal cord of the neonatal rat (Fig. 8A). A significant increase in the adenosine concentration was also evoked by isohydric hypercapnia (183.7 ± 19.0% of control, n = 6) but not isocapnic acidosis (110.4 ± 4.3% of control, n = 4).
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Discussion |
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In the rat hippocampus, hypercapnic acidosis suppresses synaptic transmission (Balestrino & Somjen, 1988; Lee et al. 1996; Velíek, 1998; Hsu et al. 2000). However, there are some inconsistent reports about the inhibitory mechanisms of hypercapnic acidosis. Hypercapnic acidosis mainly inhibits excitability of postsynaptic neurons with little or no effect on transmitter release from presynaptic neurons (Balestrino & Somjen, 1988). By contrast, it is reported that the inhibition is due to a decrease in the amplitude of presynaptic action potentials (Hsu et al. 2000). In the case of the neonatal rat spinal cord, however, hypercapnic acidosis did not inhibit the compound action potential of the dorsal root and the glutamate-evoked depolarization. Therefore, it is suggested that hypercapnic acidosis inhibits presynaptic transmitter release without affecting action potential conduction. The acidic pH is known to inhibit glutamate NMDA receptors (MacBain & Mayer, 1994). In the present experiments, unexpectedly, the response to exogenously applied glutamate was enhanced by acute hypercapnic acidosis. The mechanism of this is not clear. It has been reported that the expression pattern of NMDA receptor subunits changes during maturation in the spinal cord (Portera-Cailliau et al. 1996; Stegenga & Kalb, 2001) and that the pH sensitivity of NMDA receptors is dependent on the subunit combination (Cull-Candy et al. 2001). NMDA receptor subunits may be relatively insensitive to protons in the neonatal rat spinal cord. It seems likely that hypercapnic acidosis effectively suppresses inhibitory transmission (e.g. glycinergic or GABAergic transmission) more than excitatory transmission.
Adenosine is well known to decrease neuronal activity and produce antinociception in the spinal cord (Sawynok, 1998; Sawynok & Liu, 2003). The accumulation of adenosine also contributes to the depression of neuronal activity in the CNS during hypoxia or ischaemia (Pedata et al. 1993; Dale et al. 2000; Park et al. 2002). However, it has been reported that adenosine is not involved in the hypercapnic acidosis-induced synaptic depression in the CA1 region of rat hippocampal slices (Hsu et al. 2000). In the vascular system, on the other hand, hypercapnic acidosis induces the release of adenosine, which is involved in vasodilatation of the rat coronary (Phillis et al. 1998) and cerebral vessels (Phillis et al. 2004). In the present experiments, we found that hypercapnic acidosis increased the release of adenosine from the spinal cord and inhibited the spinal reflex potentials. In addition, this inhibitory effect was almost abolished by the increase in the external Ca2+ concentration. In rat hippocampus, it has been reported that hypoxia causes the release of adenosine and depression of synaptic transmission, which are reduced by high extracellular Ca2+ (Dale et al. 2000). High extracellular Ca2+ may also reduce the hypercapnic acidosis-evoked adenosine release and relieve the inhibition of synaptic transmission by adenosine in the spinal cord.
It has been reported that adenosine inhibits synaptic transmission of substantia gelatinosa neurons (Li & Perl, 1994; Lao et al. 2004) and reflex potentials of the isolated rat spinal cord via A1 receptors (Nakamura et al. 1997). The activation of A1 receptors is reported to suppress presynaptic transmitter release in the spinal cord (Li & Perl, 1994; Lao et al. 2004). Adenosine released by hypercapnic acidosis was also considered to inhibit transmitter release through A1 receptors, because the hypercapnic acidosis-evoked depression of the spinal reflex potentials was greatly reversed by CPT, a selective A1 receptor antagonist. Recently, it was reported that A2A receptors also caused inhibition of excitatory synaptic transmission by facilitating inhibitory transmission in the lateral horn neurons of the rat spinal cord (Brooke et al. 2004). In the present study, however, it is unlikely that A2A receptors contribute to the depression of spinal reflex responses induced by hypercapnic acidosis, because ZM243185, a selective A2A receptor antagonist, had no effect on them.
CPT, an A1 receptor antagonist, significantly but not completely reversed the hypercapnic acidosis-induced depression. In addition, both A1 and A2A receptor antagonists did not affect changes in the basal ventral root potential induced by acidosis and/or hypercapnia. The inhibitory effects of an adenosine kinase inhibitor on MSR and sVRP were completely reversed by CPT without effect on the basal ventral root potential. These results indicate that the change in the basal ventral root potential and a part of the depression of the spinal reflex potentials in response to hypercapnic acidosis are mediated by adenosine-independent mechanisms. As acidosis modulates voltage-gated Na+, K+ and Ca2+ channels (Tombaugh & Somjen, 1996) in rat hippocampal neurons, hypercapnic acidosis might directly influence such channels in the spinal cord of the neonatal rat.
In the current study, hypercapnic acidosis increased the extracellular adenosine concentration. The extracellular adenosine level is regulated by various enzymes. Released ATP is degraded to adenosine by ecto-5'-nucleotidase. Hypoxia has been reported to enhance ecto-5'-nucleotidase activity in rat aortic endothelial cells (Ledoux et al. 2003). In rat cardiomyocytes (Kitakaze et al. 1996), hypoxia-evoked adenosine release was inhibited by AOPCP, an ecto-5'-nucleotidase inhibitor. However, in the present study, AOPCP did not affect the hypercapnic acidosis-evoked depression of the spinal reflex potentials, although it increased spontaneous activities in the neonatal rat spinal cord. These results indicate that hypercapnic acidosis does not influence ecto-5'-nucleotidase activity which contributes to the maintenance of extracellular adenosine levels under normal conditions. It is also possible that the accumulation of adenosine in the extracellular space is caused by elevation of the intracellular adenosine concentration. Adenosine kinase and deaminase are key enzymes in regulating the intracellular adenosine concentration. In rat spinal cord slices, both enzyme inhibitors increase extracellular adenosine levels (Golembiowska et al. 1995). Adenosine kinase is predominantly involved in regulating adenosine levels in the rat spinal cord (Golembiowska et al. 1996) and hippocampus (Pak et al. 1994). In the present study, NH2dAD, an adenosine kinase inhibitor, depressed the spinal reflex potentials, which were reversed by CPT. On the other hand, EHNA, an adenosine deaminase inhibitor at 10 µM, a concentration sufficient to inhibit the activity of this enzyme completely, did not affect the spinal reflex responses. Higher concentrations of EHNA (50 and 100 µM) depressed sVRP but not MSR. It is unlikely that this depression is mediated by specific inhibition of adenosine deaminase, because this inhibitory effect was not reversed by CPT. These results support the idea that the inhibition of adenosine kinase results in enough extracellular adenosine accumulation to depress the spinal reflex potentials in the neonatal rat.
In our experiments, hypercapnic acidosis and isohydric hypercapnia, but not isocapnic acidosis, caused inhibition of adenosine kinase activity, release of adenosine, and depression of the spinal reflex potentials significantly reversed by CPT. It has been reported that hypercapnic acidosis causes a rapid and large fall in pHi, but isocapnic acidosis results in little pHi decrease in cultured hippocampal neurons (Bouyer et al. 2004). In rat locus coeruleus slices, hypercapnic acidosis and isohydric hypercapnia also cause a rapid fall in pHi, while isocapnic acidosis causes a much slower change in pHi (Filosa et al. 2002). Hypercapnia may rapidly change pHi, because CO2 easily enters the cells. If this is the case, it is suggested that the effects of hypercapnia are largely mediated via decreasing pHi in the isolated spinal cord of the neonatal rat. In rat hippocampal slices, it has been also reported that hypercapnic acidosis-induced suppression of synaptic transmission is mediated by changes in pHi but not pHo (Lee et al. 1996; Velíek, 1998). A low pHi during hypercapnia is suggested to inhibit the activity of adenosine kinase, because the optimum pH levels for purified adenosine kinase are 5.5 and 7.5–8.5, and its activity is suppressed at a pH around 6.7 (Yamada et al. 1980, 1981). It has been reported that hypoxia inhibits the activity of adenosine kinase via the activation of protein kinase C in the canine coronary artery (Minamino et al. 1995). Decreasing pHi may also inhibit adenosine kinase activity via the intracellular signal pathways such as protein kinase C activation. We cannot exclude the possibility that the CO2 molecule and/or bicarbonate directly cause these effects in the spinal cord. On the other hand, quite recently, it was reported that isohydric hypercapnia hardly caused depression of synaptic transmission and release of adenosine, and suggested that the effects of hypercapnic acidosis depend on pHo but not pHi in the rat hippocampal slices (Dulla et al. 2005). This discrepancy may be due to differences in the preparation used. Alternatively, isohydric hypercapnia under the modest condition (10% CO2) may not be enough to change pHi and thus to release adenosine in the hippocampal slices.
In conclusion, we have demonstrated here that acute hypercapnia potently depresses the spinal reflex potentials and that the depression is significantly reversed by an adenosine A1 receptor antagonist in the isolated spinal cord of the neonatal rat. It is suggested that a fall of pHi inhibits adenosine kinase activity and thus causes accumulation of intracellular adenosine, which results in adenosine release from the spinal cord of the neonatal rat.
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