Nanomolar ouabain augments Ca2+ signalling in rat hippocampal neurones and glia



This article is corrected by:

  1. Errata: Corrigenda Volume 591, Issue 19, 4937, Article first published online: 1 October 2013

M. P. Blaustein: Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA.   Email:

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 Ca2+ transients in neurons; Glu also evoked small, delayed transients in some astrocytes. Low-dose ATP evoked Ca2+ 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 nm ouabain (EC50 < 1 nm) augmented neuronal Glu- and CCh-evoked Ca2+ transients; this augmentation was mediated by α3 Na+ pumps and Na+–Ca2+ exchangers.
  • • Ouabain pre-incubation also augmented ATP-evoked astrocyte Ca2+ transients mediated by α2 Na+ pumps.
  • • Nanomolar ouabain and impaired α3 and α2 Na+ pump activity influence Ca2+ 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 Ca2+ 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 μm l-glutamate (Glu) and 3 μm carbachol (CCh) evoked rapid Ca2+ transients only in neurones, and small, delayed transients in some astrocytes, whereas 0.5–1 μm ATP evoked Ca2+ transients only in astrocytes. Both cell types responded to 5–10 μm Glu or ATP. The signals evoked by 3–4 μm Glu in neurones were markedly inhibited by 3–10 μm MPEP (blocks metabotropic glutamate receptor mGluR5) and 10 μm LY341495 (non-selective mGluR blocker), but not by 80 μm AP5 (NMDA receptor blocker) or by selective block of mGluR1 or mGluR2. Pre-incubation (0.5–10 min) with 1–10 nm ouabain (EC50 < 1 nm) augmented Glu- and CCh-evoked signals in neurones. This augmentation was abolished by a blocker of the Na+–Ca2+ exchanger, SEA0400 (300 nm). Ouabain (3 nm) pre-incubation also augmented 10 μm cyclopiazonic acid plus 10 mm caffeine-evoked release of Ca2+ from the neuronal endoplasmic reticulum (ER). The implication is that nanomolar ouabain inhibits α3 Na+ pumps, increases (local) intracellular Na+, and promotes Na+–Ca2+ exchanger-mediated Ca2+ gain and increased storage in the adjacent ER. Ouabain (3 nm) also increased ER Ca2+ release and enhanced 0.5 μm 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.