Stimulation of high affinity γ-aminobutyric acid_B receptors potentiates the depolarization-induced increase of intraneuronal ionized calcium content in cerebellar granule neurons

In the treatment of spasticity, the therapeutic cerebrospinal fluid levels of (±)-baclofen, a γ-aminobutyric acid (GABA)_B receptor agonist, are below 1 μM. However, the mechanism of the therapeutic action of (±)-baclofen remains unknown, because, for the most part, the action of (±)-baclofen on GABA_B receptors requires micromolar concentrations. Using fura-2 fluorescence microscopy, intracellular ionized calcium was measured in cerebellar granule neurons. Stimulation of a high affinity GABA_B receptor potentiated by 2-3-fold the rise in intracellular calcium observed after depolarization of the cell with a Krebs Ringer's buffered solution containing 40 mM K⁺. Both GABA (100 nM) and (±)-baclofen (10-100 nM) stimulated this high affinity receptor. The potentiation of the depolarization-induced rise in intracellular calcium by (±)-baclofen (100 nM) was completely blocked by the GABA_B receptor antagonist CGP 35348 (200 μM). Also, the intracellular calcium response induced by the activation of high affinity GABA_B receptors was prevented by dantrolene (10 μM). The cerebellar granule neurons contained calcium-induced calcium release (CICR) stores. Caffeine (3 mM) and ryanodine (100 μM) potentiated the depolarization-induced rise in intracellular calcium, and this response to both drugs was blocked by dantrolene (10 μM). Because dantrolene does not prevent the rise in intracellular calcium after cell depolarization (this calcium originated from the influx of extracellular calcium), (±)-baclofen acting via the high affinity GABA_B receptor indirectly activates the CICR stores, allowing the influx of extracellular calcium to trigger the release of calcium from these dantrolene-sensitive CICR stores. Thus, this high affinity GABA_B receptor might become activated during persistent depolarization caused by pathological states and could be a mechanism to be studied for the therapeutic action of (±)-baclofen in spasticity.