Glial cells have heart: rH1 Na+ channel mRNA and protein in spinal cord astrocytes.

Astrocytes in vitro express several distinct voltage-sensitive sodium currents, including tetrodotoxin (TTX)-resistant in non-stellate astrocytes and TTX-sensitive currents in stellate astrocytes. However, the molecular identity of the underlying channels, and the mechanisms that regulate their expression, have yet to be identified. Since spinal cord astrocytes in vitro express sodium currents that are nearly ten-fold greater that those of astrocytes derived from other regions, we used reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunocytochemistry to search for a sodium channel mRNA and protein corresponding to a TTX-resistant channel in these cells. RT-PCR did not detect transcripts for SNS, which is known to encode a TTX-resistant current in dorsal root ganglion neurons. However, RT-PCR demonstrated the presence of rH1 mRNA in cultured spinal cord astrocytes derived from postnatal day 0 (P0) Sprague Dawley rats at 7 days in vitro and in also intact spinal cords of P0 and P7 rats. Hybridization signal for rH1 mRNA was detected by in situ hybridization cytochemistry in most non-stellate and, at varying levels, in stellate astrocytes in these cultures. Immunocytochemical studies, utilizing a polyclonal antibody (R-12) generated against a conserved polypeptide sequence of sodium channels, demonstrated sodium channel immunoreactivity in non-stellate and stellate astrocytes in these cultures. Spinal cord cultures reacted with a rH1-specific polyclonal antibody also showed rH1 immunostaining in non-stellate and stellate astrocytes, although the intensity of the rH1 immunoreactivity in both astrocyte morphologies was attenuated compared to that observed with the R-12 generic sodium channel antibody. The presence of rH1 mRNA and protein in non-stellate astrocytes in vitro provides a possible correlate for the TTX-resistant current that has been recorded in these cells. Since TTX-resistant current is not present in stellate astrocytes, the presence of rH1 mRNA and protein in these cells suggests, in addition, that post-translational mechanisms participate in the control of sodium channel expression in these cells.

NGF has opposing effects on Na+ channel III and SNS gene expression in spinal sensory neurons.

Following sciatic nerve transection, the expression of sodium channel III (alpha-III) transcripts increases and SNS (alpha-SNS) transcripts decreases in small (< 25 microns diameter) dorsal root ganglion (DRG) neurons, which may reflect an interruption of retrograde transport of peripherally derived factor(s) involved in the regulation of these channels. To test the hypothesis that the neurotrophin nerve growth factor (NGF), which is abundant in peripheral targets, participates in the modulation of the expression of these sodium channel transcripts, we examined the hybridization signal of alpha-SNS and alpha-III mRNAs in small DRG neurons from adult rats that had been dissociated and maintained for 7 days in the absence or presence of exogenous NGF. Neurons maintained in control (no added NGF) cultures showed changes in alpha-III and alpha-SNS hybridization signal similar to those induced by axotomy, with increased alpha-III mRNA levels and decreased alpha-SNS mRNA levels, compared with those observed in small DRG neurons at 1 day in vitro. The addition of exogenous NGF to DRG cultures attenuated these alterations in transcript levels, decreasing alpha-III mRNA and increasing alpha-SNS mRNA expression. These results suggest that NGF participates in the regulation of membrane excitability in small DRG neurons by pathways that include opposing effects on different sodium channel genes.

Schwann cells modulate sodium channel expression in spinal sensory neurons in vitro.

In order to study the factors that govern the expression of sodium channel alpha-, beta1- and beta2-subunits, the influence that Schwann cells (SC) exert in the expression of sodium channels in DRG neurons was examined with in situ hybridization, immunocytochemistry, and patch clamp recording. The expression of sodium channel alpha-, beta1-, and beta2-subunit mRNAs in DRG neurons isolated from E15 rats cultured in defined medium in the absence (control) or presence of SC, or in SC-conditioned medium, was examined with isoform-specific riboprobes for sodium channel alpha-subunits I, II, III, NaG, Na6, hNE/PN1, SNS, and beta1- and beta2-subunits. DRG neurons cultured in the presence of SC displayed a significant (P < 0.05) increase in the hybridization signal for NaG, Na6, SNS, and Na beta2 mRNAs in comparison to control DRG neurons. In contrast, in SC-conditioned medium, only the hybridization signal for SNS mRNA was significantly increased. The upregulation of sodium channel mRNAs in DRG neurons co-cultured with SC was paralleled by an increase in sodium channel immunoreactivity of these cells. An increase in the mean sodium current density in DRG neurons in the presence of SC was also observed. These results demonstrate that a SC-derived factor selectively upregulates sodium channel alpha- and beta-subunit mRNAs in DRG neurons isolated from E15 rats that is reflected in an increase in functional sodium channels in these cells. This culture system may allow elucidation of the SC factor(s) that modulate the expression of sodium channels in DRG neurons.

Sodium channel mRNA in the B104 neuroblastoma cell line.

B104 neuroblastoma cells are excitable, but the ion channels underlying electrogenesis in these cells have not been identified. RT-PCR, restriction enzyme analysis and in situ hybridization were used to study sodium channel mRNAs in B104 cells. High levels of sodium channel alpha-subunit mRNAs III, NaG and Na6 and beta 1-subunit mRNA were detected by RT-PCR in B104 cells. Low levels of types I and II alpha-subunit mRNAs were also present. In situ hybridization with subtype-specific riboprobes detected sodium channel alpha-subunit mRNAs III, NaG and Na6 and beta 1-subunit mRNA in B104 cells; analysis of the percentage of B104 cells expressing each alpha-subunit mRNA subtype suggests that some cells express the mRNAs for several alpha-subunits.