Puebla M et al. (2024), Control of astrocytic Ca2+ signaling by nitric ...

XB-ART-60709

Biol Res 2024 Apr 30;571:19. doi: 10.1186/s40659-024-00503-3.

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Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels.

Puebla M , Muñoz MF , Lillo MA , Contreras JE , Figueroa XF .

Abstract
BACKGROUND: Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. RESULTS: Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. CONCLUSIONS: Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.

PubMed ID: 38689353
PMC ID: PMC11059852
Article link: Biol Res
Grant support: [+]

Genes referenced: calhm1

Article Images: [+] show captions

	Fig. 1. The Ca2+ signaling initiated by the activation of metabotropic glutamate receptors (mGluRs) in primary cultures of astrocytes relies on a cGMP-independent NO-mediated pathway. A Time course of the increase in [Ca2+]i observed in primary cultures of astrocytes in response to 10 µM glutamate before (control) and after blocking NO production with 100 µM Nω-nitro-L-arginine (L-NA). B Time course of the Ca2+ signaling evoked by the stimulation of mGluRs with t-ACPD in control conditions and in the presence of L-NA. Horizontal bars indicate the period of stimulation. C, Maximal increment in [Ca2+]i induced by glutamate in control conditions and after treating the astrocyte cultures with 10 µM ODQ, a soluble guanylyl cyclase inhibitor, or 50 µM ascorbic acid (AA), a potent reducer. Values are means ± SEM. *P < 0.05 vs Control by two-way ANOVA. †P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test
	Fig. 2. Activation of metabotropic glutamate receptors (mGluRs) in primary cultures of astrocytes evokes NO-dependent S-nitrosylation. A Immunofluorescence analysis of total protein S-nitrosylation in primary cultures of astrocytes in response to the stimulation with glutamate or t-ACPD in control conditions and after the inhibition of NO production with 100 µM Nω-nitro-L-arginine (L-NA). The effect of the vehicle of glutamate and t-ACPD is also shown. B and C Fluorescence intensity analysis of the protein S-nitrosylation observed in the experiments shown in A. Changes in fluorescence intensity are expressed in arbitrary units (A.U.). Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test
	Fig. 3. The nitric oxide synthase (NOS) isoforms endothelial (eNOS) and neuronal (nNOS), but not the inducible (iNOS), are found in astrocytes. Detection of the expression of eNOS, nNOS and iNOS (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes. The cell nuclei are highlighted by the staining with DAPI (blue)
	Fig. 4. The glutamate-initiated Ca2+ signaling in astrocytes is mediated by the opening of Cx hemichannels and Panx-1 channels. A Time course of the increase in ethidium uptake observed in primary cultures of astrocytes in response to 10 µM glutamate in control conditions and in the presence of the mimetic peptides 37,43Gap27 (100 µM) or 10Panx (100 µM) or the NOS inhibitor Nω-nitro-L-arginine (L-NA, 100 µM). The peptide 37,43Gap27 is a blocker of hemichannels formed by Cx37 or Cx43 and 10Panx is an inhibitor of the channels formed by Panx-1. B Time course of the increment in ethidium uptake attained before and after the treatment with 60 nM Nω-Propyl-L-Arginine (Nω-Propyl-L-Arg), a selective inhibitor of the neuronal nitric oxide synthase isoform. Horizontal bars indicate the period of stimulation. C Time course of the increase in [Ca2+]i induced by glutamate in control conditions and in the presence of 37,43Gap27 or 10Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test
	Fig. 5. The Cx hemichannel- and Panx-1 channel-mediated Ca2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca2+]i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM Nω-nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca2+]i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43Gap27 or 10Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. †P < 0.05 vs Baseline by paired Student’s t-test
	Fig. 6. The increase in [Ca2+]i, activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B–D Representative Western blot and densitometric analysis of CALHM1 (B), Cx43 (C) and Panx-1 (D) expression in the primary cultures of astrocytes shown in A. Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file 1: Fig. S7. E, Maximal increase in [Ca2+]i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F. Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. †P < 0.05 vs siControl by two-way ANOVA
	Fig. 7. Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM Nω-nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test
	Fig. 8. NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca2+. Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)
	Fig. 9. Schematic model of the signaling events that mediate the increase in [Ca2+]i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca2+]i by the release of Ca2+ from the intracellular Ca2+ stores through activation of an inositol (1,4,5)-triphosphate (IP3)-mediated pathway. This astrocytic Ca2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca2+ influx through these membrane channels contributes to amplify the intracellular Ca2+ store-initiated Ca2+ signaling. In addition, the increase in [Ca2+]i can be coordinated through the propagation of an inter-astrocyte Ca2+ signal via ATP release or directly by gap junction communication (GJ)

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