Stimulation of two metabotropic glutamate-receptor subtypes, mGluR1 and mGluR5, triggers the release of Ca2+ from intracellular stores through the inositol-(1,4,5) trisphosphate (InsP3) pathway. Here we report that glutamate induces single-peaked intracellular Ca2+ mobilization in mGluR1alpha-transfected cells but elicits Ca2+ oscillations in mGluR5a-transfected cells. The response patterns of the intracellular Ca2+ increase depend upon the identity of a single amino acid, aspartate (at position 854) or threonine (at position 840), located within the G-protein-interacting domains of mGluR1alpha and mGluR5a, respectively. Pharmacological and peptide mapping analyses indicated that phosphorylation of the threonine residue at position 840 of mGluR5a by protein kinase C (PKC) is responsible for the generation of Ca2+ oscillations in mGluR5a-expressing cells. To our knowledge this is the first evidence that PKC phosphorylation of G-protein-coupled receptors is important in producing oscillations in intracellular Ca2+ signalling.

The complete Freund's adjuvant (CFA)-induced arthritic rat model has extensively served as a laboratory model in the study of arthritic pain. However, the time courses of allodynia and hyperalgesia and the efficacies of different analgesics have not fully been analyzed in this model. Mechanical allodynia, thermal and joint hyperalgesia, and other disease development parameters (body weight, mobility, paw volume, and joint stiffness) were measured on postinoculation days (PIDs) 0 to 28 in rats. Acute analgesic efficacies of drugs were evaluated on PID 9 when degrees of allodynia, hyperalgesia, and joint stiffness in the ipsilateral paw reached almost the maximum, although those in the contralateral paw changed only slightly. In the ipsilateral paw, thermal hyperalgesia reached the maximum on PID 1, whereas mechanical allodynia and joint hyperalgesia progressively developed during the first 7 or 8 days, being tuned in to arthritis development. In the contralateral paw, thermal hyperalgesia never occurred, whereas mechanical allodynia and joint hyperalgesia developed after PID 11. Morphine and tramadol had full efficacies for all the pain parameters tested at sedation-inducing doses. Indomethacin and diclofenac significantly but partially improved thermal and joint hyperalgesia. Amitriptyline significantly reduced thermal and joint hyperalgesia only at sedation-inducing dose. Acetaminophen, carbamazepine, and gabapentin had, at the most, very small efficacies. In conclusion, the present study provided integrated information about the time course of pain and other disease development parameters in the CFA-induced arthritic rats, and clarified acute efficacies of different categories of analgesics for the allodynia and hyperalgesia.The complete Freund's adjuvant (CFA)-induced arthritic rat model has extensively served as a laboratory model in the study of arthritic pain. Mechanical allodynia, thermal hyperalgesia and pain on joint movement (joint hyperalgesia), which are prominent features in arthritic pain, have proved to be present in it (Tatsuo et al., 1994;Jasmin et al., 1998;Bertorelli et al., 1999). The time courses of their progression after the CFA inoculation, however, have not been fully analyzed. The first aim in the present study, therefore, was to provide integrated information about the time courses of pain (mechanical allodynia, thermal hyperalgesia, and joint hyperalgesia) and other disease development (body weight, mobility, paw volume, and joint stiffness) parameters after the CFA inoculation into the single hind paw in rats. Surprisingly, there are no studies that have fully evaluated the analgesic efficacies of different categories of analgesics on mechanical allodynia, thermal hyperalgesia, and joint hyperalgesia in the CFA-induced arthritic rat model. The second aim in the present study, therefore, was to investigate this issue. Antipyretics (e.g., acetaminophen) and nonsteroidal anti-inflammatory drugs (e.g., indomethacin and diclofenac) are the first line drugs in the treatment of arth...

(840) of mGluR5a by PKC interferes with the signal transduction through mGluR5a. We hypothesized that repetitive phosphorylation and dephosphorylation of mGluR5a could induce [Ca2ϩ] i oscillations by signaling on and off. In mGluR1␣, nonphosphorylation at aspartate (854) produces a non-oscillatory and PKC activator-resistant Ca 2ϩ response (9). This previous study provides the first evidence that an agonist can produce oscillatory/non-oscillatory patterns of Ca 2ϩ response by stimulating different receptor subtypes. However, it remained uncertain whether and how these two mGluRs control different cellular processes depending on their oscillatory/non-oscillatory Ca 2ϩ responses. We report here that prolonged stimulation of mGluR1␣ induced an increase in [Ca2ϩ] i that consisted of an initial transient peak and a subsequent steady plateau or an oscillatory increase in [Ca 2ϩ ] i . The transient phase was largely attributed to Ca 2ϩ release from intracellular Ca 2ϩ stores, but the sustained phase was solely due to Ca 2ϩ influx through a mGluR1␣ receptor-operated Ca 2ϩ channel. On the other hand, prolonged stimulation of mGluR5a continuously induced [Ca 2ϩ ] i oscillations through mobilization of Ca 2ϩ from the intracellular Ca 2ϩ stores. The coupling mechanism in the sustained phase of Ca 2ϩ response is determined by oscillatory/non-oscillatory patterns of the initial Ca 2ϩ response but not by the receptor identity. Thus, during prolonged stimulation of mGluRs, oscillatory/nonoscillatory patterns of Ca 2ϩ response lead to different coupling mechanisms in Ca 2ϩ signaling. MATERIALS AND METHODSFura-2 acetoxymethyl ester (Fura-2/AM) and phorbol-12-myristate-13-acetate (PMA) were from Wako Pure Chemical Industries. SK&F96365 and nimodipine were from Funakoshi. The mGluR1␣ antagonist, 1a-(N-phenyl)carbamoyl-1a,7a-dihydro-7(1H)-hydroxyiminocyclopropa[b]chromen (10) was synthesized in our laboratory.For construction of the mutant receptors, mGluR1␣(T) and mGluR5a(D), aspartate (854) of mGluR1␣ and threonine (840) of mGluR5a were changed into threonine and aspartate, respectively, as described previously (9). The cDNA encoding rat mGluR1␣, mGluR5a,

Metabotropic glutamate receptor type 1 (mGluR1) is thought to play important roles in the neurotransmission and pathogenesis of several neurological disorders. Here, we describe the radioligand binding properties and pharmacological effects of a newly synthesized, high-affinity, selective, and noncompetitive mGluR1 antagonist, 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a] for mGluR1 over mGluR subtypes 2 to 7, ionotropic glutamate receptors, and other receptor, transporter, and ion channel targets. In in vivo experiments, orally administered YM-298198 showed a significant analgesic effect in streptozotocin-induced hyperalgesic mice at doses (30 mg/kg) that did not cause Rotarod performance impairment, indicating that it is also useful even for in vivo experiments. In conclusion, YM-298198 is a newly synthesized, high-affinity, selective, and noncompetitive antagonist of mGluR1 that will be a useful pharmacological tool due to its highly active properties in vitro and in vivo. Its radiolabeled form [ 3 H]YM-298198 will also be a valuable tool for future investigation of the mGluR1.

Transient receptor potential vanilloid 1 (TRPV1) is activated by a variety of stimulations, such as endogenous ligands and low pH, and is believed to play a role in pain transmission. TRPV1 antagonists have been reported to be effective in several animal pain models; however, some compounds induce hyperthermia in animals and humans. We discovered the novel TRPV1 antagonist (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide (AS1928370) in our laboratory. AS1928370 bound to the resiniferatoxin-binding site on TRPV1 and inhibited capsaicinmediated inward currents with an IC 50 value of 32.5 nM. Although AS1928370 inhibited the capsaicin-induced Ca 2ϩ flux in human and rat TRPV1-expressing cells, the inhibitory effect on proton-induced Ca 2ϩ flux was extremely small. In addition, AS1928370 showed no inhibitory effects on transient receptor potential vanilloid 4, transient receptor potential ankyrin 1, and transient receptor potential melastatin 8 in concentrations up to 10 M. AS1928370 improved capsaicin-induced secondary hyperalgesia and mechanical allodynia in an L5/L6 spinal nerve ligation model in rats with respective ED 50 values of 0.17 and 0.26 mg/kg p.o. Furthermore, AS1928370 alleviated inflammatory pain in a complete Freund's adjuvant model at 10 mg/kg p.o. AS1928370 had no effect on rectal body temperature up to 10 mg/kg p.o., although a significant hypothermic effect was noted at 30 mg/kg p.o. In addition, AS1928370 showed no significant effect on motor coordination. These results suggest that blockage of the TRPV1 receptor without affecting the proton-mediated TRPV1 activation is a promising approach to treating neuropathic pain because of the potential wide safety margin against hyperthermic effects. As such, compounds such as ASP1928370 may have potential as new analgesic agents for treating neuropathic pain.

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This review focuses on the in vitro and in vivo neuropharmacology of YM872, a potential neuroprotective agent currently undergoing clinical trials in the United States (trial name: AMPA Receptor Antagonist Treatment in Ischemic Stroke -ARTIST). Its neuroprotective properties in rats and cats with induced focal cerebral ischemia are described. YM872, [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydroquinoxalin-1-yl]-acetic acid monohydrate, is a selective, potent and highly water-soluble competitive á-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. YM872 has a potent inhibitory effect on [ 3 H]AMPA binding with a K i value of 0.096 ìM. In contrast, YM872 has very low affinity for other ionotropic glutamate receptors. The solubility of YM872 is approximately 500 to 1000 times higher than that of the other competitive AMPA antagonists: YM90K, NBQX, or CNQX. The neuroprotective efficacy of YM872 was investigated in rats and cats subjected to permanent occlusion of the left middle cerebral artery. The animals were assessed either histologically or neurologically following ischemia. In rats with occluded middle cerebral artery (MCAO) YM872, by i.v. infusion, significantly reduced infarct volume measured at 24 h and 1 week after ischemia. Significant neuroprotection was maintained even when drug administration was delayed 337