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

Key points center dot Co-cultured rat hippocampal neurons and astrocytes express high-ouabain-affinity Na+ pumps with, respectively, 3 and 2 catalytic subunits. center dot 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. center dot Studies with NMDA receptors and metabotropic glutamate receptor (mGluR) blockers revealed that the neuronal Glu-evoked transients were mediated primarily by mGluR5 metabotropic receptors. center dot Pre-incubation with 110nm ouabain (EC50<1nm) augmented neuronal Glu- and CCh-evoked Ca2+ transients; this augmentation was mediated by 3 Na+ pumps and Na+Ca2+ exchangers. center dot Ouabain pre-incubation also augmented ATP-evoked astrocyte Ca2+ transients mediated by 2 Na+ pumps. center dot Nanomolar ouabain and impaired 3 and 2 Na+ pump activity influence Ca2+ signalling and may thus modulate synaptic transmission in the brain. This could explain the physiological manifestations of 3 and 2 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 neuroneastrocyte co-cultures. The neurones and astrocytes express Na+ pumps with a high-ouabain-affinity catalytic subunit (3 and 2, respectively); both also express pumps with a ouabain-resistant 1 subunit. Neurones and astrocytes were identified by immunocytochemistry and by stimulation; 34m l-glutamate (Glu) and 3m carbachol (CCh) evoked rapid Ca2+ transients only in neurones, and small, delayed transients in some astrocytes, whereas 0.51m ATP evoked Ca2+ transients only in astrocytes. Both cell types responded to 510m Glu or ATP. The signals evoked by 34m Glu in neurones were markedly inhibited by 310m MPEP (blocks metabotropic glutamate receptor mGluR5) and 10m LY341495 (non-selective mGluR blocker), but not by 80m AP5 (NMDA receptor blocker) or by selective block of mGluR1 or mGluR2. Pre-incubation (0.510min) with 110nm ouabain (EC50<1nm) augmented Glu- and CCh-evoked signals in neurones. This augmentation was abolished by a blocker of the Na+Ca2+ exchanger, SEA0400 (300nm). Ouabain (3nm) pre-incubation also augmented 10m cyclopiazonic acid plus 10mm caffeine-evoked release of Ca2+ from the neuronal endoplasmic reticulum (ER). The implication is that nanomolar ouabain inhibits 3 Na+ pumps, increases (local) intracellular Na+, and promotes Na+Ca2+ exchanger-mediated Ca2+ gain and increased storage in the adjacent ER. Ouabain (3nm) also increased ER Ca2+ release and enhanced 0.5m ATP-evoked transients in astrocytes; these effects were mediated by 2 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.