title: N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C.
abstract: 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 (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+]i). 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+]i 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 microM NMDA in the presence, but not the absence, of co-agonist (10 microM 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 microM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 microM PDBu for 1-2 min) also potentiated mechanically induced [Ca2+]i 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 microM 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.
authors.affiliation: Center for Neurovisceral Sciences and Women's Health, Department of Medicine, University of California, Warren Hall, Room 14-103, 900 Veterans Avenue, Los Angeles 90095, USA.
authors.forename: V V
authors.initials: VV
authors.lastname: Chaban
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: H S
authors.initials: HS
authors.lastname: Ennes
authors.forename: J
authors.initials: J
authors.lastname: Nie
authors.forename: E A
authors.initials: EA
authors.lastname: Mayer
authors.forename: J A
authors.initials: JA
authors.lastname: McRoberts
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000378:metabolism / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D009994:Osmolar Concentration; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010812:Physical Stimulation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D007211:Indoles; D008301:Maleimides; D016194:Receptors, N-Methyl-D-Aspartate; D015240:Phorbol 12,13-Dibutyrate; D016202:N-Methylaspartate; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D002118:Calcium