N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C

N-methyl-D-aspartate (NMDA) receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DIRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+],). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1-3 days. [Ca2+], responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2+ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 muM NMDA in the presence, but not the absence, of co-agonist (10 muM D-serine) caused transient [Ca2+](i) responses (101 9 nM) and potentiated [Ca2+](i) peak responses to subsequent mechanical stimulation more than two-fold (P<0.001). NMDA-mediated potentiation of mechanically induced [Ca2+](i) responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 muM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 muM PDBu for 1-2 min) also potentiated mechanically induced [Ca2+], responses nearly two-fold (P<0.001), while longer exposure (>10 min) inhibited the [Ca2+](i) transients by 44% (P<0.001). Both effects of PDBu were prevented by prior treatment with GFX Inhibition of voltage-dependent Ca2+ channels with 25 muM La3+ had no effect on mechanically induced [Ca2+](i) transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2+ currents by a process that was sensitive to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2+ channels which contribute to mechanically induced [Ca2+](i) transients through a PKC-mediated process. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.