Inhibitory effects of dopamine on spinal synaptic transmission via dopamine D1-like receptors in neonatal rats.

BACKGROUND AND PURPOSE: Dopamine released from the endings of descending dopaminergic nerve fibres in the spinal cord may be involved in modulating functions such as locomotion and nociception. Here, we examined the effects of dopamine on spinal synaptic transmissions in rats. EXPERIMENTAL APPROACH: Spinal reflex potentials, monosynaptic reflex potential (MSR) and slow ventral root potential (sVRP), were measured in the isolated spinal cord of the neonatal rat. Dopamine release was measured by HPLC. KEY RESULTS: Dopamine at lower concentrations (<1 µM) depressed sVRP, which is a C fibre-evoked polysynaptic response and believed to reflect nociceptive transmission. At higher concentrations (>1 µM), in addition to a potent sVRP depression, dopamine depolarized baseline potential and slightly depressed MSR. Depression of sVRP by dopamine was partially reversed by dopamine D(1) -like but not by D(2) -like receptor antagonists. SKF83959 and SKF81297, D(1) -like receptor agonists, and methamphetamine, an endogenous dopamine releaser, also caused the inhibition of sVRP. Methamphetamine also depressed MSR, which was inhibited by ketanserin, a 5-HT(2A/2C) receptor antagonist. Methamphetamine induced the release of dopamine and 5-HT from spinal cords, indicating that the release of endogenous dopamine and 5-HT depresses sVRP and MSR respectively. CONCLUSION AND IMPLICATIONS: These results suggested that dopamine at lower concentrations preferentially inhibited sVRP, which is mediated via dopamine D(1) -like and other unidentified receptors. The dopamine-evoked depression is involved in modulating the spinal functions by the descending dopaminergic pathways.

Differential contributions of adenosine to hypoxia-evoked depressions of three neuronal pathways in isolated spinal cord of neonatal rats.

BACKGROUND AND PURPOSE: Hypoxic effects on neuronal functions vary significantly with experimental conditions, but the mechanism for this is unclear. Adenosine has been reported to play a key role in depression of neuronal activities in the CNS during acute hypoxia. Hence, we examined the effect of acute hypoxia on different spinal reflex potentials and the contribution of adenosine to them. EXPERIMENTAL APPROACH: Spinal reflex potentials, monosynaptic reflex potential (MSR), slow ventral root potential (sVRP) and dorsal root potential (DRP), were measured in the isolated spinal cord of the neonatal rat. Adenosine release was measured by using enzymatic biosensors. KEY RESULTS: In the spinal cord preparation isolated from postnatal day 5-8 rats at 27°C, acute hypoxia induced adenosine release and depressed three reflex potentials. However, in postnatal day 0-3 rats at 27°C, the hypoxic-induced adenosine release and depression of MSR were negligible, while the depression of sVRP and DRP were perceptible responses. In postnatal day 0-3 rats at 33°C, hypoxia evoked adenosine release and depression of MSR. An adenosine A(1) receptor selective antagonist and a high [Ca(2+)](o), which suppressed adenosine release, abolished the hypoxic-induced depression of MSR but not those of sVRP and DRP. CONCLUSIONS AND IMPLICATIONS: Hypoxic-induced depression of MSR depends on adenosine release, which is highly susceptible to age, temperature and [Ca(2+)](o). However, a large part of the depressions of DRP and sVRP are mediated via adenosine-independent mechanisms. This differential contribution of adenosine to depression is suggested to be an important factor for the variable effects of hypoxia on neuronal functions.

Adenosine and inosine release during hypoxia in the isolated spinal cord of neonatal rats.

BACKGROUND AND PURPOSE: Adenosine and inosine accumulate extracellularly during hypoxia/ischaemia in the brain and may act as neuroprotectants. In spinal cord, there is pharmacological evidence for increases in extracellular adenosine during hypoxia, but no direct measurements of purine release. Furthermore, the efflux pathways and origin of extracellular purines are not defined. To characterize hypoxia-evoked purine accumulation, we examined the effect of acute hypoxia on the extracellular levels of adenosine and inosine in isolated spinal cords from rats. EXPERIMENTAL APPROACH: Extracellular adenosine and inosine concentrations were assayed in an in vitro preparation of the isolated spinal cord of the neonatal rat by HPLC. KEY RESULTS: The extracellular level of inosine was about 10-fold higher than that of adenosine. Acute hypoxia (10 min) caused a temperature-dependent increase in these two purines, which were inhibited by an increase in external Ca(2+), but not by several inhibitors of efflux pathways or metabolic enzymes of adenine nucleotides. Inhibitors of adenosine deaminase or the equilibrative nucleoside transporter (ENT) abolished the hypoxia-evoked increase in inosine but not adenosine. The inhibition of glial metabolism abolished the increase of both purines evoked by hypoxia but not by oxygen-glucose deprivation, hypercapnia or an adenosine kinase inhibitor. CONCLUSIONS AND IMPLICATIONS: Our data suggest that hypoxia releases adenosine itself from intracellular sources. Inosine formed intracellularly may be released through ENTs. During hypoxia, astrocytes appear to play a key role in purine release from neonatal rat spinal cord.

Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons.

In inflamed tissues, extracellular pH decreases and acidosis is an important source of pain. Histamine is released from mast cells under inflammatory conditions and evokes the pain sensation in vivo, but the cellular mechanism of histamine-induced pain has not been well understood. In the present study, we examined the effects of histamine on [Ca(2+)](i) and membrane potential responses to acid in isolated mouse dorsal root ganglion (DRG) neurons. In capsaicin-sensitive DRG neurons from wild-type mice, acid (>pH 5.0) evoked [Ca(2+)](i) increases, but not in DRG neurons from transient receptor potential V1 (TRPV1) (-/-) mice. Regardless of isolectin GS-IB4 (IB4)-staining, histamine potentiated [Ca(2+)](i) responses to acid (>or=pH 6.0) that were mediated by TRPV1 activation. Histamine increased membrane depolarization induced by acid and evoked spike discharges. RT-PCR indicated the expression of all four histamine receptors (H1R, H2R, H3R, H4R) in mouse DRG. The potentiating effect of histamine was mimicked by an H1R agonist, but not H2R-H4R agonists and was inhibited only by an H1R antagonist. Histamine failed to potentiate the [Ca(2+)](i) response to acid in the presence of inhibitors for phospholipase C (PLC) and protein kinase C (PKC). A lipoxygenase inhibitor and protein kinase A inhibitor did not affect the potentiating effects of histamine. Carrageenan and complete Freund's adjuvant produced inflammatory hyperalgesia, but these inflammatory conditions did not change the potentiating effects of histamine in DRG neurons. The present results suggest that histamine sensitizes acid-induced responses through TRPV1 activation via H1R coupled with PLC/PKC pathways, the action of which may be involved in the generation of inflammatory pain.

Roles of purines in synaptic modulation evoked by hypercapnia in isolated spinal cord of neonatal rat in vitro.

BACKGROUND AND PURPOSE: The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord. EXPERIMENTAL APPROACH: Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods. KEY RESULTS: Hypercapnia (20% CO2) depressed the reflex potentials, which were partially reversed by an adenosine A1 receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A1 receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X7 receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia. CONCLUSIONS AND IMPLICATIONS: These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A1 receptors.

Involvement of prostaglandin E(2) derived from enteric glial cells in the action of bradykinin in cultured rat myenteric neurons.

We characterized bradykinin (BK)-induced changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca(2+) imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca(2+)]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a B1 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca(2+)]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca(2+)]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E(2) (PGE(2)). The release of PGE(2) from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca(2+)]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca(2+)]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca(2+)]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus.

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons.

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.

Zinc modulates primary afferent fiber-evoked responses of ventral roots in neonatal rat spinal cord in vitro.

Zinc ions (Zn(2+)) are known to modulate the functions of a variety of channels, receptors and transporters. We examined the effects of Zn(2+) on the reflex potentials evoked by electrical stimulation and responses to depolarizing agents in the isolated spinal cord of the neonatal rat in vitro. Zn(2+) at low concentrations (0.5-2 microM) inhibited, but at high concentrations (5 and 10 microM) augmented, a slow depolarizing component (slow ventral root potential). Zn(2+) had no effect on fast components (monosynaptic reflex potential; fast polysynaptic reflex potential). Unlike Zn(2+), strychnine (5 microM), a glycine receptor antagonist, and (S),9(R)-(-)-bicuculline methobromide (10 microM), a GABA(A) receptor antagonist, potentiated both fast polysynaptic reflex potential and slow ventral root potential. Zn(2+) (5 microM) did not affect depolarizing responses to glutamate and N-methyl-D-aspartate. Zn(2+) enhanced the substance P-evoked depolarization in the absence of tetrodotoxin (0.3 microM) but not in its presence. The dorsal root potential was inhibited by (S),9(R)-(-)-bicuculline methobromide (10 microM) but not by Zn(2+) (5 microM). The Zn(2+)-potentiated slow ventral root potential was inhibited by the N-methyl-D-aspartate receptor antagonists, ketamine (10 microM) and DL-2-amino-5-phosphaonovaleric acid (50 microM) but not by P2X receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 microM) and 2',3'-O-(2,4,6-trinitrophenyl)ATP (10 microM). Ketamine (10 microM) and DL-2-amino-5-phosphaonovaleric acid (50 microM) almost abolished spontaneous activities increased by Zn(2+). It is concluded that Zn(2+) potentiated slow ventral root potential induced by primary afferent stimulation, which was mediated by the activation of N-methyl-D-aspartate receptors but not by activation of P2X receptors or blockade of glycinergic and GABAergic inhibition. Zn(2+) does not seem to directly affect N-methyl-D-aspartate receptors. The release of glutamate from interneurons may play an important role in Zn(2+)-induced potentiation of slow ventral root potential in the spinal cord of the neonatal rat.

Antiplatelet action of R-99224, an active metabolite of a novel thienopyridine-type G(i)-linked P2T antagonist, CS-747.

1. CS-747 is a novel thienopyridine-type platelet ADP inhibitor which lacks in vitro activity. This study examined pharmacological profiles of R-99224, a hepatic metabolite of CS-747. 2. R-99224 produced a concentration-dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03 - 1 microg ml(-1)), which was relatively specific to ADP compared to collagen and thrombin. 3. R-99224 (0.1 - 3 microg ml(-1)) also elicited a similar inhibition of ADP-induced aggregation in rat platelets. The inhibition by R-99224 (10 microg ml(-1)) persisted even after platelets were washed three times. Intravenous injection of R-99224 (0.1 - 3 mg kg(-1)) to rats resulted in a dose-dependent inhibition of ex vivo ADP-induced platelet aggregation. 4. R-99224 (0.1 - 100 microM) decreased binding of [(3)H]-2-methylthio-ADP ([(3)H]-2-MeS-ADP), a stable ligand for platelet ADP receptors, to washed human platelets. The inhibition by R-99224 reached a plateau at a concentration of 3 microM (1.4 microg ml(-1)), but complete inhibition was not achieved even at the highest concentration used (100 microM). 5. R-99224 (10 microM) in combination with ARL-66096 (0.3 microM), an ATP analogue-type G(i)-linked P2T receptor antagonist, produced no additional inhibition of [(3)H]-2-MeS-ADP binding. In contrast, [(3)H]-2-MeS-ADP binding was completely abolished by R-99224 (10 microM) in combination with A3P5PS (300 microM), a selective P2Y(1) antagonist, suggesting that R-99224 selectively binds to the G(i)-linked P2T receptor. 6. R-99224 (0.01 - 3 microg ml(-1)) inhibited ADP-induced [(125)I]-fibrinogen binding to human platelets in a concentration-dependent manner. R-99224 (0.1 - 1 microg ml(-1)) also inhibited the ADP-induced decrease in cyclic AMP levels in PGE(1)-stimulated platelets, whereas the agent did not affect ADP (10 microM)-induced Ca(2+) mobilization. 7. These findings suggest that R-99224 is a selective and irreversible antagonist of G(i)-linked P2T receptors and that R-99224 is a responsible molecule for in vivo actions of CS-747.

Two types of relaxation-mediating P2 receptors in rat gastric circular muscle.

Effects of purinoceptor antagonists on the relaxant responses to adenine nucleotides were examined to characterize the subtypes of P2-receptor in rat gastric circular muscle. In tissues contracted by acetylcholine, a P2-receptor antagonist, suramin (100 microM), inhibited the relaxant responses to ATP, adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) and alpha,beta-methylene ATP but not that to adenosine, while a P1-receptor antagonist, 8-phenyltheophylline (3 microM) did vice versa. The inhibitory effect of suramin was more potent for the relaxant responses to alpha,beta-methylene ATP than those to ATP or ADPbetaS. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (3-30 microM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (30 and 100 microM) inhibited the relaxation caused by alpha,beta-methylene ATP but not by ATP, ADPbetaS or adenosine. These results suggest that ATP and ADPbetaS cause relaxation via the classical P2Y receptors resistant to PPADS and DIDS. In addition, alpha,beta-methylene ATP causes relaxation via the distinct P2 receptors sensitive to PPADS and DIDS in rat gastric circular muscle.

Influence of purines and pyrimidines on circular muscle of the rat proximal stomach.

The effects of UTP were examined to characterize the receptor subtypes for UTP in the circular smooth muscle of the rat proximal stomach. The rank order of potency for contraction was 2-methylthio ATP > > ATP > or = UDP = UTP > or = adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) > > UMP > CTP = alpha,beta-methylene ATP > adenosine = uridine. In tissues contracted by acetylcholine, ATP, 2-methylthio ATP, alpha,beta-methylene ATP and adenosine each caused relaxation. alpha,beta-Methylene ATP had the most potent effect and UTP caused only a small relaxation. Suramin inhibited ATP- and UTP-induced contractions. The contractile responses to ATP decreased in tissues desensitized with UTP, ATP-gamma-S and 2-methylthio ATP, but not with alpha,beta-methylene ATP. However, UTP-induced contraction was not inhibited by desensitization with ATP, alpha,beta-methylene ATP, ATP-gamma-S and 2-methylthio ATP. These results suggest that UTP causes contraction via receptors different from common P2 purinoceptors. These receptors are blocked by suramin in the rat proximal stomach.

Modulation of calcium current by ATP in guinea-pig adrenal chromaffin cells.

The effects of extracellular adenosine 5'-triphosphate (ATP) on voltage-dependent Ca2+ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 microM) reversibly suppressed Ca2+ currents in the presence of 5mM Ca2+ in the extracellular solution. The inhibitory effect of ATP on Ca2+ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10mM EGTA, on the other hand, the inhibitory effect did not change regardless of the amplitude of current responses to ATP. In the presence of 10 mM Ba2+, ATP (100 microM) reduced Ba2+ currents in a manner similar to Ca2+ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]; 1 mM) or guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma-S]; 100 microM). A depolarizing prepulse to + 100 mV partly relieved ATP-induced reduction of Ba2+ currents. ADP, AMP and adenosine also reduced Ba2+ currents and the effect of adenosine was the most potent. Adenosine (0.5 and 1 mM) significantly inhibited adrenaline secretion induced by nicotine (50 microM). These results suggest that Ca2+ entry through ATP-activated non-selective cation channels results in the inactivation of voltage-dependent Ca2+ channels. In addition, ATP seems to modulate Ca2+ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.

Muscarinic receptor subtypes mediating catecholamine secretion and increase in intracellular Ca2+ concentration in adrenal chromaffin cells of the guinea pig.

Muscarinic receptor subtypes mediating catecholamine secretion and increase in the intracellular concentration of Ca2+ ([Ca2+]i) were examined using muscarinic agonists and antagonists in dispersed adrenal chromaffin cells of the guinea pig. All muscarinic agonists (1-1,000 microM) tested caused increases in adrenaline secretion in a dose-dependent manner. Muscarine and methacholine were more effective than bethanechol, oxotremorine and pilocarpine. Muscarine and oxotremorine caused a small increase in adrenaline secretion even in the absence of extracellular Ca2+. Both 4-DAMP (0.1 microM) and pirenzepine (0.1 microM), but not methoctramine (0.1 microM), shifted the dose-response curve for muscarine-induced adrenaline secretion to the right. These muscarinic agonists also caused increase in [Ca2+]i in the presence of extracellular Ca2+. Muscarine-induced [Ca2+]i rises were reduced, but not abolished, by removal of extracellular Ca2+. These results suggest that catecholamine secretion induced by muscarinic agonists is mediated through M1, or M1 and M3 muscarinic receptor subtypes in adrenal chromaffin cells of the guinea pig.

Characteristics of ATP-induced catecholamine secretion from adrenal chromaffin cells of the guinea-pig.

The effect of ATP on catecholamine secretion and intracellular Ca2+ concentration ([Ca2+]i) was examined using perfused adrenal glands and dispersed chromaffin cells of the guinea-pig. The application of ATP (2 to 10 mM) for 2 min caused a dose-dependent increase in catecholamine secretion from perfused adrenal glands. ADP, but neither AMP nor adenosine, was also effective in increasing catecholamine secretion, though its potency was much less than that of ATP. 3. The ATP-induced secretory response was observed even under Na(+)-deficient conditions, but was reversibly abolished by removal of extracellular Ca2+. 4. In dispersed chromaffin cells, ATP (0.5 mM) caused increases in catecholamine secretion and [Ca2+]i, both of which were abolished after the removal of extracellular Ca2+. 5. These results indicate that ATP released from adrenal chromaffin cells together with catecholamine may exert a positive feedback influence on the following exocytotic catecholamine secretion. This effect may be accomplished by increasing the entry of extracellular Ca2+, but not by mobilizing intracellular Ca2+ stores in adrenal chromaffin cells of the guinea-pig.

ATP-evoked membrane current in guinea pig adrenal chromaffin cells.

Adenosine 5'-triphosphate (ATP) evoked an inward current in whole-cell voltage-clamped adrenal chromaffin cells of the guinea pig. The reversal potential (Erev) of ATP-evoked current was about 0 mV in normal external solution and was shifted towards negative potentials by substituting Tris+ or sucrose, but not Ca2+, for the extracellular Na+. This current was mediated by the activation of non-selective cation channels and had some different properties from nicotinic current. It is suggested that these channels may function as a part of the ATP-induced Ca2+ influx pathway in guinea pig chromaffin cells.