Expression of voltage‐gated Ca²⁺ channel subtypes in cultured astrocytes

Abstract

Transient intracellular [Ca 2ϩ ] increases in astrocytes from influx and/or release from internal stores can release glutamate and thereby modulate synaptic transmission in adjacent neurons. Electrophysiological studies have shown that cultured astrocytes express voltage-dependent Ca 2ϩ channels but their molecular identities have remained unexplored. We therefore performed RT-PCR analysis with primers directed to different voltage-gated Ca 2ϩ channel ␣ 1 subunits. In primary cultures of astrocytes, we detected mRNA transcripts for the, and ␣ 1G (T-type), but not ␣ 1A (P/Q-type), voltage-gated Ca 2ϩ channels. We then used antibodies against all of the Ca 2ϩ channel subunits to confirm protein expression, via Western blots, and localization by means of immunocytochemistry. In Western blot analysis, we observed immunoreactive bands corresponding to the appropriate ␣ 1 subunit proteins. Western blots showed an expression pattern similar to PCR results in that we detected proteins for the ␣ 1B (N-type), ␣ 1C (L-type), ␣ 1D (L-type), ␣ 1E (R-type), and ␣ 1G (T-type), but not ␣ 1A (P/Q-type). Using immunocytochemistry, we observed Ca 2ϩ channel expression for these subunits in punctate clusters on plasmamembrane of GFAP-expressing astrocytes. These results confirm that cultured astrocytes express corresponding proteins to several high-and low-threshold Ca 2ϩ channels but not ␣ 1A (P/Q-type). Overall, our data indicate that astrocytes express multiple types of voltage-gated Ca 2ϩ channels, hinting at a complex regulation of Ca 2ϩ homeostasis in glial cells. GLIA 41:347-353, 2003.

Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes

“…We used synaptosomal plasma membrane-enriched preparation (LP-1) as a positive control. Immunoblot analysis revealed the absence of a neuron-specific protein, SNAP-25 (Parpura et al, 1995b;Latour et al, 2003), in astrocytic culture, although SNAP-25 was reliably detected in LP-1 preparations. Next, we performed RT-PCR using mRNA isolated from rat brain and from astrocytic cultures (Fig.…”

Hypersensitive Glutamate Signaling Correlates with the Development of Late-Onset Behavioral Morbidity in Diffuse Brain-Injured Circuitry

“…The remaining KCl-evoked glutamate release after conotoxin application may represent a pool of conotoxin-insensitive glutamate release, with the conotoxin-sensitive pool vulnerable to diffuse TBI. However, astrocyte modulation of neuronal release cannot be completely ruled out, as N-type calcium channels (blocked by x-conotoxin) are also present on a small subset of astrocytes in the CNS (Latour et al, 2003). Regardless, the injuryinduced increase in the sub-second evoked release of glutamate was likely of neuronal origin.…”

P2X2receptors are essential for [Ca2+]iincreases in response to ATP in cultured rat myenteric neurons

“…It has been reported that voltage-dependent Ca 2ϩ channels (VDCCs) are primarily expressed in neurons (27). However, some glial cells also express VDCCs (40). Nicotinic ACh receptors are reported to be expressed in myenteric neurons (38,65).…”

“…The high-K ϩ -induced [Ca 2ϩ ] i response has been used to discriminate neurons from other cells in guinea pig myenteric neurons (14,56). However, it has been reported that some glial cells possess VDCCs (40). Nicotinic ACh receptors are known to be expressed in myenteric neurons (38,65).…”