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Ammonium increases Ca(2+) signalling and upregulates expression of Cav1.2 gene in astrocytes in primary cultures and in the in vivo brain

Ammonium increases Ca(2+) signalling and upregulates expression of Cav1.2 gene in astrocytes in primary cultures and in the in vivo brain

Acta Physiologica 214(2): 261-274

The primary aim of this study was to identify the effects of hyperammonaemia on functional expression of Cav1.2 L-type Ca(2+) channels in astroglia. Primary cultures of mouse astrocytes were used to study effects of chronic treatment (1-5 days) with ammonium chloride, at 1, 3 and 5 mm on depolarization-induced increases in free cytosolic Ca(2+) concentration ([Ca(2+)]i , measured with Fura-2 based microfluorimetry) in control conditions and following treatment with the L-type Ca(2+) channel inhibitor, nifedipine, or with ryanodine receptor inhibitor, ryanodine. Expression of Cav1.2 mRNA was identified with RT-PCR, whereas protein content was determined by Western blotting. Sustained hyperammonaemia in vivo was induced by daily injections of urease (33 units kg body weight(-1), i.p.) for 3 days. Depolarization-induced [Ca(2+)]i transients sensitive to nifedipine (peak of the response) and to ryanodine (plateau phase) were significantly increased in astrocytes chronically exposed to ammonium. The ammonium-induced increase in Ca(2+) influx in astrocytes resulted from an upregulation of Cav1.2 channel's expression detected at mRNA and protein levels. Increase in Cav1.2 expression was prevented by ouabain antagonist canrenone. Similar upregulation of Cav1.2 gene expression was found in the brains of adult mice subjected to intraperitoneal injection of urease. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers and treated with intraperitoneal injections of urease, the fluorescence-activated cell sorting of neurones and astrocytes demonstrated that Cav1.2 mRNA expression was upregulated in astrocytes, but not in neurones. Ammonium-induced deregulation of astroglial Ca(2+) signalling, is, in part, associated with upregulation of Cav1.2 L-type calcium channels.

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Accession: 057168346

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PMID: 25846713

DOI: 10.1111/apha.12500

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