Brain-type Natriuretic Peptide Regulation And Mechanisms Of The Nervous System, Potassium Channel

In addition to natriuretic peptieds'roles in cardiovascular control and fluid volume regulation,a number of studies have revealed that NPs can modulate membrane ion channels.Brain natriuretic peptide may act as a neuromodulator via its associated receptors in the nervous system.However,few studies have reported on its activity on ion channels in the nervous system.In this study,we chose two models of neurons and glia cells to study the effect of BNP on K⁺ channels which are among the most diverse families of membrane proteins.Schwann cells are the satellite cell of the peripheral nervous system, responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K⁺ currents,including inactivating A-type currents(I_A), delayed-rectifier currents(I_K),and inward-rectifier currents(K_IR),constitute the main conductances found in SCs.Physiological studies have shown that I_K channels may play an important role in SC proliferation.Cerebellar granule neurons are widely used as a model to study neuronal fucntions.Primary cerebellar granule cells have been shown to possess various outward K_V currents,including outward transient K⁺ current (I_A),outward rectifier delayed K⁺ current(I_K),and the non-inactivating K⁺ current I_K(so).Here,we used the methods of patch-clamp recordings,immunocytochemistry and confocal microscopy,cell proliferation assays,DNA constructs and cell transfection to investigate the role of BNP in voltage-gated K⁺ currents and detailed subunits in the nervous system.The research is divided into three parts as followings.Part One:In this study,we determined that BNP upregulated the TEA-sensitive delayed rectifier outward potassium current(I_K) in mouse Schwann cells using whole-cell recording techniques.At concentrations of 1-500 nM,BNP reversibly activated I_K in a dose-dependent manner,with modulating its steady-state activation and inactivation properties.The effect of BNP on I_K was abolished by pre-incubation with the specific antagonist of NPR-A,and could not be mimicked by application of the NPR-C agonist.These results were supported by immunocytochemical findings indicating that NPR-A was abundantly expressed in Schwann cells.The application of 8-Br-cGMP mimicked the effect of BNP on I_K,but BNP was unable to further increase I_K after the application of 8-Br-cGMP.The effect of BNP on I_K was blocked by KT5823,an inhibitor of cGMP-dependent protein kinase(PKG).Genistein blocked I_K and also completely eliminated the effects of BNP and 8-Br-cGMP on I_K. Physiological studies have shown that I_K channels may play an important role in SC proliferation.BNP significantly stimulated Schwann cell proliferation and this effect could be partly inhibited by TEA.We therefore concluded that BNP modulated I_K via cGMP-and tyrosine kinase-dependent pathways by activation of NPR-A.This effect of BNP on I_K in Schwann cells might be involved in its effect on cell proliferation.Part Two:In this study,we determined that BNP suppressed the TEA-sensitive delayed rectifier outward potassium current(I_K) in rat cerebellar granular cells using whole-cell recording techniques.At concentrations of 10-1000 nM,BNP reversibly activated I_K in a dose-dependent manner,with modulating its steady-inactivation properties.The effect of BNP on I_K was abolished by pre-incubation with the specific antagonist of NPR-A,and could not be mimicked by application of the NPR-C agonist.The application of 8-Br-cGMP mimicked the effect of BNP on I_K,but BNP was unable to further increase I_K after the application of 8-Br-cGMP.The effect of BNP on I_K was blocked by KT5823,an inhibitor of cGMP-dependent protein kinase (PKG).Moreover,incubation of isolated cell cultures in the BNP-containing medium also increased cGMP innunoreactivity in the granule cells.We therefore concluded that BNP modulated I_K via cGMP-PKG signal pathway by the activation of NPR-A.Part Three:The effect of BNP on the amplitude of I_K was opposite on the Schwann cells and cerebellar granule cells,even through the similar signal pathway. This might result from different subunits regulated by BNP.Since Kv2.1 was the main a subunit of I_K on granule cells,we apply JZTX-â…¢,the specific blocker of Kv2.1,to inhibit Kv2.1αsubunit and then the effect of BNP on I_K was disappeared on granule cells.It seems that BNP effect Kv2.1αsubunit on granule cells.Besides,on the HEK293 cells transfected with pmCherry-Kv2.1,BNP could modulate the steady-inactivation property,similar to the modulation on native cerebellar granule cells.However,with the presence of JZTX-â…¢,the dominant effect of BNP could still be observed on Schwann cells,indicating that BNP might regulate other subunits on Schwann cells as well as Kv2.1αsubunit.Thus,we investigated the effect of BNP on the HEK293 cells transfected with pmCherry-Kv1.2 or pEGFP-Kv1.5.BNP could up-regulate the amplitude of Kv1.2αsubunit and down-regulate the amplitude of Kv1.5αsubunit.BNP could also modulate steady-state activation and inactivation properties of Kv1.2αsubunit,similar to the results observed in native Schwann cells.