In normal rat forebrain, the NR1/NR2A and NR1/NR2B dimmers are the main constitutional forms of NMDA receptors. The present study was carried out to determine the functional properties of the heteromeric NMDA receptor subunits and their inhibition by bis(7)-tacrine (B7T). Rat NR1, NR2A and NR2B cDNAs were transfected into human embryonic kidney 293 cells (HEK-293).The inhibition of NMDA-activated currents by B7T was detected in HEK-293 cell expressing NR1/NR2A or NR1/NR2B receptors by using whole-cell patch-clamp techniques. The results showed that in HEK-293 cells expressing NR1/NR2A receptor, 1μmol/L B7T inhibited 30μmol/L NMDA- and 1000μmol/L NMDA-activated steady-state currents by 46% and 40%, respectively (P>0.05; n=5), suggesting that the inhibition of B7T on NR1/NR2A receptor doesn't depend on NMDA concentration, which is consistent with a non-competitive mechanism of inhibition. But for the NR1/NR2B receptor, 1μmol/L B7T inhibited 30μmol/L NMDA- and 1000 μmol/L NMDA-activated steady-state currents by 61% and 13%, re-spectively (P<0.05; n=6), showing that B7T appears to be competitive with NMDA. In addition, simultaneous application of 1μmol/L B7T and 1000μmol/L NMDA produced a moderate inhibition of peak NMDA-activated current, followed by a gradual decline of the current to a steady state. However, the gradual onset of inhibition produced by B7T applied simultaneously with NMDA was eliminated when B7T was given 5s before NMDA. These results suggested that B7T inhibition of NMDA current mediated by NR1/NR2B receptor was slow onset, and it did not depend on the presence of the agonist. With holding potentials ranging from -50 to +50 mV, the B7T inhibition rate of NMDA currents didn't change significantly, and neither did the reversal potential. We are led to conclude that the NR1/NR2B recombinant receptor can serve as a very useful model for studying the molecular mechanism of NMDA receptor inhibition by B7T.
·AIM:To investigate whether bis(7)-tacrine,a multifun-ctional drug,inhibits N-methyl-D-aspartate (NMDA)-activated current in retinal ganglion cells(RGC) and provides neuroprotection against retinal cell damage.·METHODS:Purified RGC cultures were obtained from retinas of 1-3 days old Sprague-Dawley(SD) rats,following a two-step immunopanning procedure.After 7 days of cultivation,the inhibition of NMDA-activated current by bis(7)-tacrine was measured by using patch-clamp recording techniques.In animal experiments,RGCs were damaged after intravitreal injection of NMDA (5|ìL,40nmol) in adult rats.Bis(7)-tacrine(0.05,0.1,0.2mg/kg) or memantine(20mg/kg) was intraperitoneal administered to the rats fifteenminutes before intravitreally injection of NMDA.RGC damage was analyzed by histologic techniques,TUNEL and retrograde labeling techniques.·RESULTS:Whole-cell patch-clamp recordings demonstrated that NMDA (30|ìmol/L) resulted in approximately-50 pA inward currents that were blocked by bis(7)-tacrine(1|ìmol/L).Histological examination and retrograde labeling analysis revealed that bis(7)-tacrine induced a significant neuroprotective effect against NMDA-induced cell damage 7 days after NMDA injection.TUNEL staining showed that pretreatment with bis(7)-tacrine was effective in ameliorating NMDA-induced apoptotic cell loss in the retinal ganglion cell layer 18 hours after injection.·CONCLUSION:Bis(7)-tacrine possesses remarkable neur-oprotective activities against retinal excitotoxicity through inhibition of NMDA receptors.·
We developed a method that allows us to label nociceptive neurons innervating tooth-pulp in rat trigeminal ganglion neurons using a retrograde fluorescence-tracing method, to record ATP-activated current in freshly isolated fluorescence-labeled neurons and to conduct single cell immunohistochemical staining for P2X1 and P2X3 subunits in the same neuron. Three types of ATP-activated current in these neurons (F, I and S) were recorded. The cells exhibiting the type F current mainly showed positive staining for P2X3 , but negative staining for P2X1 . The results provide direct and convincing evidence at the level of single native nociceptive neurons for correlation of the characteristics of ATP-activated currents with their composition of P2X1 and P2X3 subunits and cell size. The results also suggest that the P2X3 , but not P2X1 , is the main subunit that mediates the fast ATP-activated current in nociceptive neurons.
