Gap junctional communication and pharmacological heterogeneity in astrocytes cultured from the rat striatum

Abstract

Indo-1 and fluo-3 imaging techniques were used to investigate the role of gap junctions in the changes in cytosolic calcium concentrations ([Ca2+]i) induced by several receptor agonists. Subpopulations of confluent cultured astrocytes from the rat striatum were superfused with submaximal concentrations of endothelin-1 (Et1) and the alpha 1-adrenergic and muscarinic receptor agonists, methoxamine and carbachol, respectively. 2. Combined binding and autoradiographic studies indicated that all striatal astrocytes possess binding sites for Et1. In contrast, alpha 1-adrenergic and muscarinic binding sites were found to be heterogeneously distributed. In agreement with these findings, Et1 induced fast calcium responses in all cells while only subsets of striatal astrocytes responded to the application of methoxamine or carbachol. 3. Halothane, heptanol and octanol, which are commonly used as gap junction inhibitors, drastically reduced the amplitude of Et1-induced calcium responses. In contrast, 18-alpha-glycyrrhetinic acid (alpha GA) used at a concentration known to block gap junction permeability in astrocytes had no significant effect on the amplitude of these calcium responses. 4. As demonstrated by quantitative and topological analysis, Et1 application similarly increased [Ca2+]i levels in all astrocytes in both the absence and presence of alpha GA. 5. In control conditions, subpopulations of cells responding to methoxamine or carbachol exhibited two main types of calcium responses which differed in their shape and kinetic characteristics. In the presence of alpha GA the number of cells responding to these receptor agonists was significantly reduced. Indeed, responses characterized by their long latency, slow rise time and weak amplitude disappeared in the presence of alpha GA while responses with short latency and fast rise time were preserved. 6. These results indicate that permeable gap junction channels tend to attenuate the pharmacological and functional heterogeneity of populations of astrocytes, while their inhibition restricts calcium responses in astrocytes expressing high densities of transmitter receptors coupled to phospholipase C.

Figure 1. Autoradiographic visualization of the distribution of specific binding sites for Et, muscarinic and α1-adrenergic receptor ligands in confluent cultures of rat striatal astrocytes

A, immunostaining with GFAP antibodies of 3-week-old astrocytes showing typical primary cultures used for autoradiographic binding assays. B-D, labelling of cultured astrocytes with [¹²⁵I] Et (B), [³H] QNB (C) and [¹²⁵I] HEAT-2 (D) indicating that binding sites for the endothelin receptor ligand are homogeneously distributed, while binding sites for muscarinic and α1-adrenergic receptor ligands are heterogeneously distributed in subsets of astrocytes and often observed in clusters (arrows). Insets, curves showing total (▪), non-specific (•) and specific (▵) binding of [¹²⁵I] Et, [³H] QNB and [¹²⁵I] HEAT-2 ligands which were used at concentrations of 1.6, 4.4 and 6.1 pM and either the presence or absence of Et1 (0.1 μM), atropine (0.1 mM) and prazosin (1 μM), respectively. Calibration bars indicate 100 μm in A and 120 μm in B (applies also to C and D).

Figure 2. Pattern of calcium responses induced by Et1 and methoxamine in cultured striatal astrocytes

A, quantification of [Ca²⁺]_i responses evoked by a brief application of 0.1 μM Et1 (horizontal bar) recorded in 5 astrocytes loaded with indo-1 AM which were selected from the same microscopic field. Note that all cells responded with similar kinetics and amplitudes. B, diversity of [Ca²⁺]_i responses induced by a brief application of 0.1 mM methoxamine in 4 astrocytes from the same microscopic field. Note that the latency, rise time and amplitude of the responses are different for each responding cell.

Figure 5. Effect of αGA on calcium responses induced by Et1 and carbachol

[Ca²⁺]_i increases evoked by a short application (horizontal bar) of 0.1 μM Et1 (A) or 1 mM carbachol (B) recorded in astrocytes loaded with indo-1 AM. C and D, responses to the application of the same receptor agonists, respectively, recorded from astrocytes which have been previously treated for 5 min with 10 μM αGA. As shown in A, note that all cells responded with similar kinetics and amplitudes to application of Et1 (C), while delayed and slow increases were suppressed when carbachol-induced increases in [Ca²⁺]_i were monitored in the presence of the uncoupler (D). In all cases, cells were recorded from the same microscopic field and originated from the same culture.

Figure 3. Cellular distribution of calcium responses induced by methoxamine superfusion of a population of confluent striatal astrocytes

A-C, pseudocolour sequence of images showing the changes in [Ca²⁺]_i evoked in the same field of astrocytes loaded with fluo-3 AM. A, basal [Ca²⁺]_i monitored 1 min before the first application of 0.1 mM methoxamine, this image was taken as the background fluorescence of the investigated microscopic field and was subtracted from images obtained thereafter. B, increase in [Ca²⁺]_i monitored 4 s after the beginning of the first brief (10 s) superfusion with 0.1 mM methoxamine. C, distribution of [Ca²⁺]_i responses monitored 3 s after the beginning of the second methoxamine superfusion performed 13 min after the first (shown in B). D, drawing representing the areas in which changes in [Ca²⁺]_i occurred in the course of the 2 successive methoxamine superfusions as shown in B and C. Shaded areas illustrate the localization of overlapping subsets of responding astrocytes (calibration bar, 200 μm).

Figure 4. Comparative effects of 18-α-glycyrrhetinic acid (αGA) and other commonly used uncoupling agents on basal [Ca²⁺]_i and on the amplitude of calcium responses induced by Et1

A-C, typical calcium responses induced by 0.1 μM Et1 in control conditions (A) and in the presence of either 10 μM αGA (B) or 2 mM halothane (C). All traces were averaged from 7 individual recordings performed in the same microscopic field of astrocytes loaded with indo-1 AM. D, histogram illustrating the basal [Ca²⁺]_i levels (left-hand side) and Et1-evoked increases in [Ca²⁺]_i (right-hand side). αGA had no effect on the Et1-evoked increase in [Ca²⁺]_i whereas in the presence of 2 mM halothane, 0.6 mM octanol or 1 mM heptanol the Et1-evoked increases in [Ca²⁺]_i were drastically reduced. In contrast, none of these uncouplers have any significant effect on the basal [Ca²⁺]_i. Data are averaged from 36–279 cells. Statistical analysis was conducted by one-way ANOVA, followed by post hoc Dunnett's multiple comparison test. Significance was established at P < 0.01 (**), all other data were not significantly different (P > 0.5). In D the vertical scale refers to relative changes in the fluorescence ratios (ΔF₄₀₅/F₄₈₀) of indo-1 emissions compared with the basal level.

Figure 6. Quantitative analysis of calcium responses to Et1, carbachol and methoxamine recorded from communicating and non-communicating confluent striatal astrocytes

Receptor agonist-induced calcium responses were plotted using 3 parameters: amplitude, latency and rise time of the increases in [Ca²⁺]_i. These responses were recorded from astrocytes perfused with 0.1 μM Et1 (A and D), 1 mM carbachol (B and E) and 0.1 mM methoxamine (C and F), in either the absence (A-C) or presence (D-F) of 10 μM αGA. The vertical scale refers to relative changes in the fluorescence ratios (ΔF₄₀₅/F₄₈₀) of indo-1 emissions compared with the basal level.

Figure 7. Selective suppression of 1 class of methoxamine- and carbachol-evoked calcium responses following inhibition of gap junction permeability

Calcium responses to the perfusion of the 3 receptor agonists were monitored in astrocytes loaded with indo-1 AM. When no significant change in [Ca²⁺]_i (relative change in fluorescence ratio < 0.05) was recorded, astrocytes were considered as non-responding cells (None). Calcium responses were separated into 2 groups depending on the kinetics of the change in [Ca²⁺]_i: ‘primary’ responses were defined by a latency shorter than 2 s and a rise time faster than 3 s while these values were higher in secondary responses. This classification was performed in either the absence (A) or presence (B) of 10 μM αGA. The analysis of these 2 types of response indicated that the inhibition of gap junctional communication results in the selective disappearance of ‘secondary’ responses which were only monitored following applications of carbachol and methoxamine. Histograms were established from a total number of 910 and 763 measurements in A and B, respectively.