Adenosine A1 receptors (A1R) are able to form a heteromeric complex with P2Y1 receptors (P2Y1R) that generates A1R with P2Y1R-like agonistic pharmacology. A potent P2Y1R agonist, adenosine 5'-O-(2-thiotriphosphate), binds the A1R binding pocket of the A1R/P2Y1R complex and inhibits adenylyl cyclase activity via Gi/o protein. These mechanisms might be used to fine-tune purinergic inhibition locally at sites where there is a particular oligomerization structure between purinergic receptors and explain the undefined purinergic functions by adenosine and adenine nucleotides.
Extracellular ATP is now recognized as a neurotransmitter or neuromodilator in the nervous system, producing diverse physiological effects by activating multiple P2 receptors. Although P2-receptor signaling is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, such a metabolic step leads to adenosine generation, thereby initiating adenosine (P1)-receptor activation. Because most cells and tissues co-express P1 and P2 receptors, ecto-nucleotidase on target tissues, especially enzymes catalyzing adenosine formation, are determinants of the cellular response to ATP. Ecto-5'-nucleotidase (E-5'-NT) has been considered to play a principal role in conversion of AMP to adenosine. In addition to E-5'-NT, we have recently demonstrated that ecto-alkaline phosphatase is also involved in ATP-induced P1-receptor activation through a rapid and localized adenosine production on the membrane surface. In this minireview, we describe the pharmacological profile of ecto-nucleotidase-dependent P1-receptor activation by ATP and molecular bases of preferential delivery of metabolically generated adenosine to P1 receptors. Several lines of evidence suggest that the close association between ecto-nucleotidases and P1 receptors may constitute a functional receptor for extracellular ATP, and some physiological responses to ATP would occur through this mechanism.
Adenosine enhanced intracellular Ca2+ concentrations in astrocytes via A2a adenosine receptors involving protein kinase A (PKA) activation. The Ca2+ rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP3-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A2a adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A2a adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca2+ release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes.
Application of 10 μM ATP for 10 min transiently depressed, then slowly augmented, synaptic transmission in CA1 neurons, leading to long-term potentiation (LTP) (ATP-induced LTP). This ATP-induced LTP was blocked by addition of an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, D,L-2-amino-5-phosphonovalerate (5 μM). For ATP-induced LTP, delivery of test synaptic inputs once every 20 s to CA1 neurons could be substituted by application of 100 nM NMDA during ATP perfusion. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 nM), whereas a P2X purinoceptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (50 μM), or a P2Y purinoceptor antagonist, basilen blue (10 μM), had no effect. These results, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors / Ca2+ channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor / Ca2+ channel.
The studies aiming to understand the function of purinoceptors in the central nervous system (CNS), which has been explored mostly in isolated and cultured cell systems, are now at the stage of identifying their physiological and pathophysiological significance in the native organs, tissues, and whole animals. The results of our recent studies made in brain slice preparations are not in full accordance with what have been demonstrated in isolated cells, mostly due to strong interplay between ATP receptors, adenosine receptors, and ecto-nucleotidases. This suggests that these proteins form coordinated regulation systems in the native tissue, controlling the local network behaviors through regulating the balance between the effects of ATP and adenosine on synaptic transmissions. We propose that this tripartite regulation system by extracellular purines may be an important target of CNS drugs.
We have been studying the role of ATP receptors in pain and already reported that activation of P2X2/3 heteromeric channel/receptor in primary sensory neurons causes acutely tactile allodynia, one hallmark of neuropathic pain. We report here that tactile allodynia under the chronic pain state requires an activation of the P2X4 ionotropic ATP receptor and p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. Two weeks after L5 spinal nerve injury, rats displayed a marked mechanical allodynia. In the rats, activated microglia were detected in the injured side of the dorsal horn and the level of the dually-phosphorylated active form of p38MAPK (phospho-p38MAPK) in these microglia was increased. Moreover, intraspinal administration of a p38MAPK inhibitor, SB203580, suppressed the allodynia. We also found that the expression level of P2X4 was increased strikingly in spinal cord microgila after nerve injury and that pharmacological blockade or inhibition of the expression of P2X4 reversed the allodynia. Taken together, our results demonstrate that activation of P2X4 or p38MAPK in spinal cord microglia is necessary for tactile allodynia after nerve injury.
Mechanisms underlying bradykinin-induced vasoconstriction were investigated in rat perfused mesenteric vascular beds with active tone. In preparations with intact endothelium, bolus injections of bradykinin (1 to 1,000 pmol) dose-dependently produced three-phase vascular effects, which consisted of a first-phase vasodilation followed by a second-phase vasoconstriction and a subsequent third-phase vasodilation; these effects were abolished by FR172357 (bradykinin B2-receptor antagonist), but not by des-Arg9-[Leu8]-bradykinin (bradykinin B1-receptor antagonist). In preparations with intact endothelium, indomethacin (cyclooxygenase inhibitor), seratrodast (thromboxane A2 (TXA2)-receptor antagonist), ONO-3708 (TXA2/prostaglandin H2 (PGH2)-receptor antagonist) or ozagrel (TXA2 synthesis inhibitor) markedly inhibited the bradykinin-induced vasoconstriction. In preparations without endothelium, the bradykinin-induced vasoconstriction was abolished by indomethacin and ONO-3708, while seratrodast and ozagrel had no effect. These results suggest that the endothelium-dependent vasoconstriction of bradykinin is mainly mediated by TXA2 and that prostanoids other than TXA2, probably PGH2, in mesenteric vascular smooth muscle are responsible for bradykinin-induced endothelium-independent vasoconstriction.
Oxybutynin has been used for neurogenic bladder disorders and is known to have anti-cholinergic and antispasmodic properties. However, the anti-cholinergic and antispasmodic properties of 4-ethylamino-2-butynyl(2-cyclohexyl-2-phenyl)glycolate hydrochloride (N-desethyloxybutynin: DEOB), a metabolite of oxybutynin, have not been clarified. Therefore, in the present study, we studied these properties by using rat urinary bladder specimens in comparison with oxybutynin. Moreover, the effect of DEOB on rhythmic urinary bladder contraction was also evaluated using anesthetized rats. DEOB and oxybutynin concentration-dependently inhibited the carbachol-induced contraction, the pA2 values being 7.19 and 7.11, respectively. DEOB and oxybutynin also concentration-dependently inhibited the 100 mM KCl-induced contraction, the ED50 values being 12.1 and 10.4 μM, respectively. Intravenously administered DEOB and oxybutynin dose-dependently (0.03 – 0.3 mg/kg) inhibited the amplitude of the rhythmic bladder contraction to similar degrees, but had no affect on the frequency. From the above results, it was determined that DEOB has anti-cholinergic and antispasmodic properties and that these activities were almost equal to those of oxybutynin. Therefore, DEOB may play an important role during oxybutynin therapy for neurogenic bladder disorder.
This study was undertaken to identify novel approaches to pharmacological treatment of asthma. Here we hypothesize that the platelet-activating factor receptor antagonist ginkgolide B (GB) in combination with the antioxidant carotenoid astaxanthin (ASX) suppresses T cell activation comparably to two commonly-used antihistamines: cetirizine dihydrochloride (CTZ) and azelastine (AZE). Peripheral blood mononuclear cells from asthmatics, cultured 24 h with either 50 μg/ml phytohemaglutinin (PHA) or PHA plus selected dosages of each drug are analyzed by flow cytometry for CD25+ or HLA-DR+ on CD3+ (T cells). Results are reported as stimulation indices (SI) of %CD3+CD25+ cells or %CD3+HLA-DR+ cells in cultures treated with PHA alone versus these subpopulations in cultures treated with both PHA and drugs. Combinations of ASX and GB exhibited optimal suppression at 10−7 M GB + 10−8 M ASX for CD3+CD25+ (SI = 0.79 ± 0.04, P = 0.001) and 10−7 M GB + 10−7 M ASX for CD3+HLA-DR+ (SI = 0.82 ± 0.05, P = 0.004). In conclusion, suppression of T cell activation below fully stimulated values by GB, ASX, and their combinations was comparable and for some combinations better than that mediated by CTZ and AZE. These results suggest that ASX and GB may have application as novel antiasthmatic formulations.
The novel antiepileptic drug gabapentin was designed as a structural analog of γ-aminobutyric acid (GABA). However, its mechanism of action remains unclear. In the present study, we investigated the effect of gabapentin on spinal reflexes in anesthetized rats. The mono- and polysynaptic reflex potentials were recorded from the ipsilateral L5 ventral root after stimulation of the L5 dorsal root. The dorsal root reflex potential, an index of presynaptic inhibition, was recorded from the ipsilateral L4 dorsal root. In non-spinalized (intact) and spinalized rats, intravenously administered gabapentin reduced the mono- and polysynaptic reflex potentials in a dose-dependent manner. These inhibitory effects of gabapentin were not suppressed by the GABAA antagonist picrotoxin. Moreover, gabapentin also decreased spinal reflexes in spinalized rats depleted of spinal GABA with semicarbazide, an inhibitor of the GABA-synthesizing enzyme. The dorsal root reflex potentials were not affected by gabapentin. These results suggest that endogenous GABA does not mediate the inhibitory effects of gabapentin on spinal reflexes.
The mechanisms of prokinetic action of tegaserod in the gastrointestinal tract has not been studied in detail. The aim of this study was to investigate the effect of tegaserod on peristaltic reflexes and propagating peristaltic waves in guinea pig ileum. A partitioned organ bath divided into three chambers was used to investigate the effect of tegaserod on peristaltic reflexes. A sensory stimulus was applied to the intermediate chamber, and changes in the circular muscle tension were monitored in a peripheral chamber. Another peristaltic bath was used to investigate the effect of tegaserod on peristaltic waves induced by intraluminal perfusion. Guinea pig ileum exhibited contractions in the circular muscle both orally and anally in response to mucosal stroking. Tegaserod (10−8 – 10−6 M) did not influence the maximal amplitude and the area under the curve of contraction both orally and anally to a mucosal stimulus. Intraluminal perfusion of fluid containing tegaserod (10−8 – 10−6 M) significantly increased the number of peristaltic waves in a concentration-dependent manner (P<0.05). Also, tegaserod (10−8 – 10−6 M) significantly increased the area under the curve of peristaltic waves (P<0.05). It is concluded that tegaserod has prokinetic action on guinea pig ileum by increasing the number of the circular muscle contractions during peristalsis.
We reported previously that intracerebroventricular (i.c.v.) administration of P2X-receptor agonists produced antinociception and the effect was attenuated by i.c.v. pretreatment with β2-adrenergic receptor antagonists. The present study examined the involvement of noradrenergic neurons arising from the locus coeruleus (LC) in the supraspinal antinociception by the P2X-receptor agonist α,β-methylene-ATP in rats. We found that pretreatment with DSP-4 (50 mg/kg, i.p.), which is a neurotoxin to selectively disrupt noradrenergic neurons arising from the LC, significantly attenuated the antinociception by i.c.v. administration of α,β-methylene-ATP (10 nmol/rat). Microinjection of α,β-methylene-ATP (0.1 and 1 nmol/side) into the bilateral LC significantly elevated the nociceptive threshold more potently than the i.c.v. administration at a dose of 10 nmol/rat. The antinociception by intra-LC injection of α,β-methylene-ATP (1 nmol/side) was significantly attenuated by co-injection of pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (1 nmol/side), a non-selective P2X-receptor antagonist. These results suggest that noradrenergic neurons arising from the LC are involved in the supraspinal antinociception by α,β-methylene-ATP through P2X receptors in the LC.
Antidepressant drugs, especially tricyclics have been widely used in the treatment of chronic pain, but not in acute pain. Because of numerous undesirable side effects, the selective serotonin reuptake inhibitors (SSRIs), with their favorable side effect profile, are preferred nowadays. An activation of the endogenous opioid mechanisms or potentiation of the analgesic effect mediated by serotonergic and/or noradrenergic pathways are thought to be involved in the antinociceptive action of SSRIs. In this study, the potential antinociceptive effect of paroxetine and its interaction with opioidergic system and serotonin receptors were evaluated. The antinociceptive effect of paroxetine was tested using a hot plate test in mice. Paroxetine, a SSRI antidepressant drug, induced an antinociceptive effect following i.p. administration. This antinociception was significantly inhibited by naloxone, an opioid receptor antagonist, suggesting the involvement of opioidergic mechanisms. While ondansetron (a 5-HT3-receptor antagonist) inhibited the effect of paroxetine, ketanserin (a 5-HT2-receptor antagonist) could not. In conclusion, paroxetine-induced antinociception, similar to morphine, suggests an involvement of direct or indirect action (via an increase in release of endogenous opioid peptide(s)) at opioid receptor sites and an involvement of serotonergic mechanisms mainly at the receptor level.
Natural products regulate cell growth in response to oncogene activation that induces cell cycle arrest and apoptosis in tumor cell lines. We investigated the mechanisms of caspase activation in human malignant melanoma, A375-S2 cells, by the natural product shikonin, which was isolated from the plant Lithospermum erythrorhizon SIEB. et ZUCC. Shikonin inhibited cell growth in a time- and dose-dependent manner, which might be mediated through up-regulation of p53 and down-regulation of cyclin-dependent protein kinase 4. Caspase activation was detected in shikonin-induced cell apoptosis, which involved in a post-mitochondrial caspase-9-dependent pathway. Decreased Bcl-2 protein levels and increased Bax protein levels were positively correlated with elevated expression of p53 protein. Apoptosis-inducing factor, another apoptotic protein of mitochondria, partially contributed to shikonin-induced release of cytochrome c. Taken together, shikonin-induced DNA damage activates p53 and caspase-9 pathways.
The methanol extract of Caesalpinia bonducella FLEMING (Caesalpiniaceae) leaves (MECB) were evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. The extract was administered at the doses of 50, 100, and 200 mg/kg body weight per day for 14 days after 24 h of tumor inoculation. After the last dose and 18 h fasting, the mice were sacrificed. The present study deals with the effect of MECB on the growth of transplantable murine tumor, life span of EAC-bearing hosts, hematological profile, and biochemical parameters such as lipid peroxidation (LPO), glutathione content (GSH), superoxide dismutase (SOD), and catalase (CAT) activities. MECB caused significant (P<0.01) decrease in tumor volume, packed cell volume, and viable cell count; and it prolonged the life span of EAC-tumor bearing mice. Hematological profile converted to more or less normal levels in extract-treated mice. MECB significantly (P<0.05) decreased the levels of lipid peroxidation and significantly (P<0.05) increased the levels of GSH, SOD, and CAT. The MECB was found to be devoid of conspicuous short-term toxicity in the mice when administered daily (i.p.) for 14 days at the doses of 50, 100, 200, and 300 mg/kg. The treated mice showed conspicuous toxic symptoms only at 300 mg/kg. The results indicate that MECB exhibited significant antitumor and antioxidant activity in EAC-bearing mice.
Sairei-to (TJ-114), a Japanese traditional medicine, has been used clinically for the treatment of various edematous disorders. The inhibitory effect on edema may be dependent on the diuretic response to TJ-114. This study was conducted to clarify the mechanism of diuresis. Pentobarbital-anesthetized rats were infused with a saline solution intravenously (0.4 mL/30 min). Urine was collected through a bladder cannula for 30 min. Intraduodenal administration of TJ-114 (0.5 – 1.5 g/kg) resulted in a dose-dependent increase of urine volume with insignificant urinary sodium excretion and significant urea excretion, but no effect on mean arterial blood pressure. Furthermore, TJ-114 significantly increased urinary levels of NO2 + NO3. In addition, intraperitoneal pre-treatment with 6 mg/kg of NG-nitro-L-arginine methyl ester inhibited the increase in urine volume, urinary urea excretion, and urinary levels of NO2 + NO3 in the rats treated with TJ-114. These results suggest that TJ-114 induces a diuretic response via production of NO.
Both the hyperthermia and augmented glutamate release in the organum vasculosum laminae terminalis (OVLT) after an intravenous dose (30 ng/kg) of staphylococcal enterotoxin A (SEA) were significantly reduced by pretreatment with intravenous administration of cyclooxygenase inhibitors such as aspirin (1 – 10 mg/kg), sodium salicylate (1 – 10 mg/kg), or diclofenac (10 mg/kg). Intra-OVLT administration of 50 – 200 μg in 1.0 μl of either aspirin or sodium salicylate 60 min before or 120 min after an intra-OVLT dose (50 μg in 1.0 μl) of glutamate also significantly suppressed the glutamate-induced hyperthermia. These findings suggest that inhibition of cyclooxygenase receptor mechanisms suppresses SEA fever by inhibition of glutamate release in the OVLT of rabbit brain.
We have elucidated the interactions of human and rat organic anion transporters (hOATs and rOATs) with pravastatin and cimetidine. Pravastatin inhibited hOAT1/rOAT1, hOAT2/rOAT2, hOAT3/rOAT3, and hOAT4. The mode of inhibition was noncompetitive for hOAT1 and hOAT2, whereas it was competitive for hOAT3 and hOAT4. Cimetidine also inhibited hOAT1/rOAT1, hOAT3/rOAT3, and hOAT4. The mode of inhibition was a combination of competitive and noncompetitive manners for hOAT1, whereas it was competitive for hOAT3. The effects of OAT inhibitors on OAT1, OAT2, and OAT3 exhibited some but not so remarkable interspecies differences between humans and rats. In conclusion, we have characterized pravastatin and cimetidine as OAT inhibitors.
Microglia are macrophage-like phagocytic cells in the brain parenchyma. However, microglial function after neurodegeneration is not fully understood. In this study, occlusion of the middle cerebral artery (MCA) and reperfusion caused massive neuronal loss in the rat cerebral cortex and striatum after 3 days. When exogenous microglia were microinjected into the intracerebroventricle during MCA occlusion, neurodegenerative areas significantly decreased. At that time, migrated microglia were detected in the ischemic lesion. These results suggest that exogenous microglia can migrate into brain parenchyma and then protect against neurodegeneration induced by MCA occlusion and reperfusion.
Unsei-in inhibits substance P (SP)-induced scratching of mice after repeated administration. The involvement of cutaneous nitric oxide (NO) in the SP-induced scratching led us to investigate the effects of Unsei-in on the cutaneous NO system in mice. Seven-day oral administration of Unsei-in (300, but not 100, mg/kg daily) significantly inhibited scratching and the increase of cutaneous NO after intradermal SP injection. The NO synthase 1 (NOS1) inhibitor 7-nitroindazole (1 nmol/site) decreased SP-induced scratching and NO production. Repeated administration of Unsei-in (300 mg/kg) reduced the cutaneous NOS1 level. The results suggest that the inhibition of cutaneous NOS1 expression and NO production participates in the antipruritic action of Unsei-in.
Characterization of the GABAB receptor in the human colon was performed by the reverse transcription-polymerase chain reaction (RT-PCR). mRNAs for both subunits of the GABAB receptor, GABAB1 and GABAB2, were detected in the human colon. The GABAB1(e) isoform was detected in the human colon, but not in the brain, and the other isoforms, except GABAB1(d), were detected in both tissues. Thus, the GABAB receptor may be present as a heterodimer with subunits of GABAB1 and GABAB2 in the human colon.