No abstract

factors and/or extracellular signalling molecules different from those controlling the astrocyte properties in the fully developed brain. The expression of voltage-dependent calcium (Ca 2+) currents and inward-rectifying potassium (K +) currents can be triggered by the co-cultivation of astrocytes with neurons [4,6]. Ca 2÷ currents were also recorded in pure astrocyte cultures after a short time elevation of the intracellular content of cAMP [7,8]. Long-term treatment with dibutyryl-cyclic-AMP (dBcAMP), a permeable analog of cAMP, of astrocyte cultures has been reported to induce morphological and biochemical changes which have also been taken as an indicator for astrocyte differentiation [9 16]. However, little is known whether in astrocytes this goes along with a parallel modification of the electric membrane properties [17].We investigated this issue in patch-clamp experiments performed on rat cultured cortical astrocytes which had been incubated for 1 3 weeks with 250HM dBcAMP. The results indicate that the prolonged strengthening of the cAMP signalling causes, in conjunction with morphological/biochemical signs of astrocyte differentiation, the new expression of kinetically and pharmacologically distinct inwardly rectifying K + and CI-conductances which may be implicated in the astrocyte function of extracellular K + buffering.

The biophysical and pharmacological properties of the inwardly rectifying Cl− conductance (IClh), expressed in rat type‐1 neocortical cultured astrocytes upon a long‐term treatment (1–3 weeks) with dibutyryl‐cyclic‐AMP (dBcAMP), were investigated with the whole‐cell patch‐clamp technique. Using intra‐ and extra‐cellular solutions with symmetrical high Cl− content and with the monovalent cations replaced with N‐methyl‐D‐glucamine, time‐ and voltage‐dependent Cl− currents were elicited in response to hyperpolarizing voltage steps from a holding potential of 0 mV. The inward currents activated slowly and did not display any time‐dependent inactivation. The rising phase of the current traces was best fitted with two exponential components whose time constants decreased with larger hyperpolarization. The steady‐state activation of IClh was well described by a single Boltzmann function with a half‐maximal activation potential at −62 mV and a slope of 19 mV that yields to an apparent gating charge of 1.3. The anion selectivity sequence was Cl− = Br− = I− > F− > cyclamate ≥ gluconate. External application of the putative Cl− channel blockers 4,4 diisothiocyanatostilbene‐2,2 disulphonic acid or 4‐acetamido‐4‐isothiocyanatostilbene‐2,2‐disulphonic acid did not affect IClh. By contrast, anthracene‐9‐carboxylic acid, as well as Cd2+ and Zn2+, inhibited, albeit with different potencies, the Cl− current. Taken together, these results indicate that dBcAMP‐treated cultured rat cortical astrocytes express a Cl− inward rectifier, which exhibits similar but not identical features compared with those of the cloned and heterologously expressed hyperpolarization‐activated Cl− channel ClC‐2. GLIA 21:217–227, 1997. © 1997 Wiley‐Liss, Inc.

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl -current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl -current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long-term effects on both neurones and astroglial cells. Whether Guo also modulates the activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 lM) promotes the functional expression of an inward rectifier K + (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up-regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo-induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo-induced up-regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that downregulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K + homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K + -buffering impairment of the astroglial syncytium that occurs in pathological conditions.