Ca²⁺/CaMK? Mediates Androgen's Rapid Effects On Synaptic Protein PSD95 In HT22 Cells

Androgen,as an important steroid hormone in the human body,is involved in many developmental and physiological processes.The classic way of action of androgens is through binding with the Androgen Receptor?AR?in the cytoplasm to form a hormone-receptor complex,which is then transported to the nucleus and interacts with the response elements on DNA.This pathway mediates the transcriptional regulation of target genes.This pathway works slowly and takes a long time.In addition,research confirms that androgens can also rapidly activate intracellular signal transduction molecules and exert related effects through AR-independent pathways.This effect is characterized by its fast speed,which is usually completed within seconds to minutes,and it does not rely on AR-mediated transcription-translation processes to directly produce corresponding effects.For example,androgens can rapidly increase the Ca²⁺concentration in distal tubules,immune T cells,and human coronary endothelial cells.This phenomenon is called the non-genomic effect of androgens.As one of the ubiquitous second messengers,Ca²⁺can widely regulate intracellular effects,including cell proliferation,apoptosis,cell migration,and gene expression.Studies have confirmed that Ca²⁺is involved in long-term potentiation?LTP?and long-term depression?LTD?phenomena.LTP and LTD are considered to be the biological basis of synaptic plasticity that is closely related to learning and memory.This project will use mouse hippocampal neuron cell line HT22 cells as a cell research model to investigate whether Ca²⁺/CaMK? mediates androgen nongenomic effects on synaptic plasticity through molecular biology,morphology,and electrophysiological techniques.Objective:To study the effect of testosterone on Ca²⁺in hippocampal neuron cell line HT22 and its mechanism;to investigate whether the Ca²⁺/CaMK? signaling pathway mediates androgen non-genomic effects of testosterone promoting the expression of synaptic protein PSD95.Methods:The intracellular Ca²⁺concentration was observed using calcium imaging and confocal microscopy;the membrane potential of HT22cells was measured by electrophysiological techniques;the expression of PSD95 was observed by immunoblotting and immunofluorescence techniques.Results:1. Effect of Testosterone on Ca²⁺in Hippocampal Neuron Cell Line HT22and Its Mechanism?1?Different concentrations of testosterone were used to intervene in HT22 cells of hippocampal neuron cell line.The results of calcium imaging HT22 cells was time-dependent and dose-effective.Internal Ca²⁺levels have the highest effect.Therefore,in the subsequent experiments,we selected 100n M testosterone and incubated for 20 min as the experimental treatment conditions.?2?The effect of testosterone on the membrane resting potential of nerve cells was tested by patch clamp technique;by incubation with L-type calcium channel blocker amlodipine or N-type calcium channel blocker tauromycin,the observation results showed L/N type voltage Gated calcium channels are involved in testosterone-induced Ca²⁺increase in hippocampal neuron cell line HT22 cells.?3?The effect of testosterone on the membrane resting potential of nerve cells was tested by patch clamp technique;by incubation with L-type calcium channel blocker amlodipine or N-type calcium channel blocker tauromycin,the observation results showed L/N type voltage Gated calcium channels are involved in testosterone-induced Ca²⁺increase in hippocampal neuron cell line HT22 cells.2.To investigate whether the Ca²⁺/CaMK? signaling pathway mediates the androgenic non-genomic effect of testosterone on promoting the expression of synaptic protein PSD95.?1?Western Blot and immunofluorescence staining methods were found to significantly inhibit testosterone on PSD95 protein levels after pretreatment with L-type calcium channel blocker amlodipine or N-type calcium channel blocker aspirin?pretreatment?.?2?Western Blot and immunofluorescence staining experiments showed that the stimulation of CaMK? protein inhibitor KN-93 in HT22 cells significantly inhibited the effect of testosterone on phosphorylated CaMK? protein,while the total CaMK? protein was not significantly different in each group.?3?Western Blot and immunofluorescence staining experiments showed that the stimulation of the CaMK? protein inhibitor KN-93 in HT22 cells significantly inhibited the effect of testosterone on PSD95 protein.Conclusions:1.Incubation of 100 n M testosterone for 20 min has a significant effect2. Increased intracellular Ca²⁺is an external calcium influx mediated by L/N voltage-gated channels on the membrane.3. Testosterone promotes the expression of phosphorylated CaMK? protein by increasing external calcium influx.4. Testosterone can promote the expression of PSD95 protein through the Ca²⁺/CaMK? pathway.