Intracellular Na⁺and metabolic modulation of Na/K pump and excitability in the rat suprachiasmatic nucleus neurons

Abstract

Na/K pump activity and metabolic rate are both higher during the day in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na(+) and energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of extracellular K(+) to block the Na/K pump excited SCN neurons to fire at higher rates and return to normal K(+) to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the presence of tetrodotoxin to block the action potentials, both zero K(+)-induced depolarization and rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric Na(+) imaging with a Na(+)-sensitive fluorescent dye indicated saturating accumulation of intracellular Na(+) in response to pump blockade with zero K(+). The Na(+) ionophore monensin also induced Na(+) loading and hyperpolarized the membrane potential, with the hyperpolarizing effect of monensin abolished in zero Na(+) or by pump blockade. Conversely, Na(+) depletion with Na(+)-free pipette solution depolarized membrane potential but retained residual Na/K pump activity. Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting potential and increase spontaneous firing in most cells, and to raise intracellular Na(+) levels in all cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative phosphorylation and glycolysis in fueling the Na/K pump. Together, the results indicate the importance of intracellular Na(+) and energy metabolism in regulating Na/K pump activity as well as neuronal excitability in the SCN neurons.

Role of Na+/Ca2+exchanger in Ca2+homeostasis in rat suprachiasmatic nucleus neurons

“…The surrounding plots of F340/ F380 (Fig. 1, A-H (Wang et al 2012b;Wang and Huang 2006). The result is consistent with previous findings that basal [Ca 2ϩ ] i in rat SCN neurons is dependent on action potentialmediated Ca 2ϩ influx (Colwell 2000;Irwin and Allen 2009).…”

“…The experiment began with the application of K ϩ -free solution to determine the condition of reduced preparations and to confirm that the cells being recorded were indeed neurons. The removal of extracellular K ϩ has been shown to block the Na ϩ -K ϩ pump to depolarize the membrane potential and increase spontaneous firing of SCN neurons, followed by rebound hyperpolarization and inhibition of spontaneous firing on return to normal K ϩ (Wang et al 2012b;Wang and Huang 2006). Figure 1 shows the effects of K ϩ -free (0 K ϩ ) and Na ϩ -free (0 Na ϩ ; Li ϩ substituted) solution on levels of [Ca 2ϩ ] i in the SCN neurons in a reduced preparation.…”

“…Figure 3A shows the increase in [Na ϩ ] i in response to 1 M monensin (n ϭ 10 cells). For comparison, K ϩ -free solution was also applied for 2 min to transiently block the Na ϩ -K ϩ pump to increase [Na ϩ ] i (see also Wang et al 2012b). Figure 3B shows a typical experiment to demonstrate the ability of 0 Na ϩ to uptake Ca 2ϩ in elevated [Na ϩ ] i with monensin (n ϭ 9 cells).…”

“…The SCN neurons of the reduced preparation could be identified visually with an inverted microscope (Olympus IX70 and IX71). The preparation thus obtained allows rapid application of drugs (Chen et al 2009) and has been used for fluorescent Na ϩ imaging (Wang et al 2012b) and to demonstrate diurnal rhythms in both spontaneous firing and Na ϩ -K ϩ pump activity (Wang and Huang 2004).…”

“…On one hand, replacement of Na ϩ with Li ϩ has complex actions on spontaneous firing to alter basal [Ca 2ϩ ] i , thus rendering it impossible to distinguish its effect on forward NCX from that on membrane potential and spontaneous firing. On the other hand, replacement of Na ϩ with NMDG ϩ lowers basal [Ca 2ϩ ] i by blocking the generation of action potentials and also hyperpolarizing the membrane potential to ϳϪ80 mV (Wang et al 2012b), which would completely close both the high-and low-threshold voltage-dependent Ca 2ϩ channels (Huang 1993) to prevent Ca 2ϩ entry via these channels. Future studies with cells clamped at resting potentials may help better resolve this issue.…”