Nanomolar ouabain augments Ca 2+ signalling in rat hippocampal neurones and glia

Key points•  Co‐cultured rat hippocampal neurons and astrocytes express high‐ouabain‐affinity Na + pumps with, respectively, α3 and α2 catalytic subunits. •  Low‐dose l ‐glutamate (Glu) and carbachol (CCh) evoked Ca 2+ transients in neurons; Glu also evoked small, delayed transients in some astrocytes. Low‐dose ATP evoked Ca 2+ transients only in astrocytes. •  Studies with NMDA receptors and metabotropic glutamate receptor (mGluR) blockers revealed that the neuronal Glu‐evoked transients were mediated primarily by mGluR5 metabotropic receptors. •  Pre‐incubation with 1–10 n m ouabain (EC 50  < 1 n m ) augmented neuronal Glu‐ and CCh‐evoked Ca 2+ transients; this augmentation was mediated by α3 Na + pumps and Na + –Ca 2+ exchangers. •  Ouabain pre‐incubation also augmented ATP‐evoked astrocyte Ca 2+ transients mediated by α2 Na + pumps. •  Nanomolar ouabain and impaired α3 and α2 Na + pump activity influence Ca 2+ signalling and may thus modulate synaptic transmission in the brain. This could explain the physiological manifestations of α3 and α2 pump mutations and certain mood disorders linked to altered Na + pump function.Abstract  Linkage of certain neurological diseases to Na + pump mutations and some mood disorders to altered Na + pump function has renewed interest in brain Na + pumps. We tested nanomolar ouabain on Ca 2+ signalling (fura‐2) in rat hippocampal neurone–astrocyte co‐cultures. The neurones and astrocytes express Na + pumps with a high‐ouabain‐affinity catalytic subunit (α3 and α2, respectively); both also express pumps with a ouabain‐resistant α1 subunit. Neurones and astrocytes were identified by immunocytochemistry and by stimulation; 3–4 μ m l ‐glutamate (Glu) and 3 μ m carbachol (CCh) evoked rapid Ca 2+ transients only in neurones, and small, delayed transients in some astrocytes, whereas 0.5–1 μ m ATP evoked Ca 2+ transients only in astrocytes. Both cell types responded to 5–10 μ m Glu or ATP. The signals evoked by 3–4 μ m Glu in neurones were markedly inhibited by 3–10 μ m MPEP (blocks metabotropic glutamate receptor mGluR5) and 10 μ m LY341495 (non‐selective mGluR blocker), but not by 80 μ m AP5 (NMDA receptor blocker) or by selective block of mGluR1 or mGluR2. Pre‐incubation (0.5–10 min) with 1–10 n m ouabain (EC 50  < 1 n m ) augmented Glu‐ and CCh‐evoked signals in neurones. This augmentation was abolished by a blocker of the Na + –Ca 2+ exchanger, SEA0400 (300 n m ). Ouabain (3 n m ) pre‐incubation also augmented 10 μ m cyclopiazonic acid plus 10 m m caffeine‐evoked release of Ca 2+ from the neuronal endoplasmic reticulum (ER). The implication is that nanomolar ouabain inhibits α3 Na + pumps, increases (local) intracellular Na + , and promotes Na + –Ca 2+ exchanger‐mediated Ca 2+ gain and increased storage in the adjacent ER. Ouabain (3 n m ) also increased ER Ca 2+ release and enhanced 0.5 μ m ATP‐evoked transients in astrocytes; these effects were mediated by α2 Na + pumps. Thus, nanomolar ouabain may strongly influence synaptic transmission in the brain as a result of its actions on the high‐ouabain‐affinity Na + pumps in both neurones and astrocytes. The significance of these effects is heightened by the evidence that ouabain is endogenous in mammals.