Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia
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Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca2+ concentration [Ca2+]i through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca2+]i oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin–Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca2+]i increases in renal epithelia. Spontaneous [Ca2+]i increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 ± 6.7% (n = 23) of the cells showed spontaneous [Ca2+]i increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca2+]i oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca2+]i oscillatory activity. The association between spontaneous [Ca2+]i increases and nucleotide signalling was further tested in 132–1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca2+]i increases. Transfection with either hP2Y6 or hP2Y2 receptors revealed a striking degree of oscillations. Similar spontaneous [Ca2+]i increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y2 knock out mice (0.050 ± 0.020 events per second, n = 8) compared to the wild type (0.147 ± 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca2+]i oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.
KeywordsSpontaneous Ca2+ oscillations Nucleotide release ATP Thick ascending limb MDCK Tubular perfusion
We thank the following foundations for their support: The Danish Medical Research Foundation. Grundforskningsfonden. Nyreforeningens forskningsfond, The Aarhus University Research Foundation, Eva og Henry Frænkels Mindefond, The A.P. Møller Foundation for the Advancement of Medical Science and R.I.E. König-Petersen Forskningsfond for Nyresygdomme. The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). We thank Jean-Marie Boeynaems, Universite Libre de Bruxelles, Belgium, and Robert Nicholas, University of North Carolina, NC, USA, for the kind provision of P2-receptor transfected cells.
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