The Effects Of Fentanyl And Combined With Propofol Or Midazolum On GABA_A Receptor In Hippocampal Pyramidal Neurons Of The Rats

Background: The actions of intravenous anesthetics and opioids have been studied for many years. Unfortunately, there was still little research works on the mechanism of their combination although the combined anesthesia has been used widely in clinical practice. GABA is the major inhibitory neurotransmitter in CNS. GABA_A receptor is thought to be the site of action for a variety of drugs with hypnotic, anesthetic and anticonvulsant properties. These drugs bind to the distinct sites on GABA_A receptors and enhance GABA_A receptors-activated Cl^- flux. Phosphorylation is an important regulatory mechanism of biomembrane ion channel. GABA_A receptors can be phosphorylated by several protein kinases, including protein kinase A (PKA) and C (PKC), calcium and calmodulin-dependent kinase â…¡ (CaMK â…¡) and protein tyrosine kinase (PTK). The effects of opioids are mediated by opioid receptors through inhibition of Ca²⁺ channel or activation of K⁺ conductance. Recently, it was found that opioids and their receptors exist widely in GABAergic pathway and exert some functions in this location. Opioids can excite CNS neurons by disinhibition, i.e. the inhibition of GABA release. Besides, the activation of ^-opioid receptor can potentiate GABA_A receptor-activated Cl" current (GABA current, Igaba) through the mediation of PKA inhibition. The fact that both intravenous anesthetics and opioids can influence the function of GABA_A receptor suggests a possible interaction existed. The hippocampus modulates memory formation, cognition and emotion, participates in general anesthesia and is rich in GABA_A receptors, opioids and their receptors. In the present study, we examined â‘ the characteristics of Igaba in acutely dissociated hippocampal pyramidal neurons of neonatal rats; â‘¡the modulatory effects of a μ-opioid receptor agonist fentanyl on the GABA response; â‘¢ the roles of PKA in the opioid modulation of Igaba and â‘£the effects of fentanyl combined with propofol or midazolum on these GABA_A receptors.Methods: The hippocampal pyramidal neurons were acutely isolated from 3- to 12-days old Sprague Dawley rats by an enzymatic-mechanic method. I_GABA was recorded with voltage-clamped, whole-cell patch-clamp condition in gap-free mode atthe holding potential (V_H) of-50 mV. The resistance between the recording electrode filled with the internal pipette solution and the reference electrode was 2～5 MΩ. A high-resistance seal (>1 GΩ) was obtained, and the negative pressure was increased to rupture the cell membrane, achieving the whole-cell configuration. Series resistance and whole-cell capacitance were adjusted and continually compensated to 70%～90% during the experiment. Data were collected with a system consisting of Axopatch 200B patch-clamp amplifier, Pentium III computer, Digidata 1200 interface and pCLAMP software. Drugs were administered by perfusion to the neurons through a gravity-driven 'Y' system. I_GABA was recorded before, after drug perfusion and after washing out. Time course of drugs was also recorded. All experiments were performed at room temperature (22～25℃). Four to twelve neurons were recorded for each treatment. â‘ The effects of fentanyl on I_GABA were studied by the GABA response to a series concentration of fentanyl from 0.00001μM to 10.0μM. After the effects of fentanyl on I_GABA reached maximal, a nonspecific opioid receptor antagonist naloxone or a μ-opioid receptor-selective antagonist CTAP was applied to test the changes of fentanyl effect. â‘¡A membrane- permeable activator of adenylate cyclase forskolin or a membrane- permeable specific PKA inhibitor H-89 was applied to assess the action of PKA on I_GABA. After I_GABA was fully modulated by PKA intervention, fentanyl combined with forskolin or H-89 was administered to examine the effects of PKA on fentanyl. â‘¢When propofol or midazolum-induced potentiation of I_GABA was studied, propofol or midazolum was applied for 8 s before a 15-s coapplication of modulator and GABA. After the effects of fentanyl on I_GABA reached maximal, propofol or midazolum was applied to examine their effects on I_GABA. Data were presented as mean±standard deviation. Student's t test and one-way ANOVA were used for statistical analyses. P values less than 0.05 were considered significant.Results:â‘ 0.00001 ～ 10.0μM fentanyl depressed I_GABA dose-dependently. Fentanyl 1.0μM shortened the desensitization time constant of I_GABA from 10974.42±2216.68ms to 3061.29±223.37ms (P<0.05). The inhibitory effects of fentanyl were reduced or prevented by naloxone or CTAP. CTAP increased the EC₅₀ of fentanyl from 0.011μM to 0.414μM (P<0.05). Fentanyl and CTAP did not change the reversal potential of I_GABA(E_Cl-=-3.0mV). Heterogeneity of GABA_A receptors was suggested by their different responses to fentanyl: I_GABA was inhibited in 79.4%, enhanced in 14.3% and not affected in 6.3 % of the studied cells.â‘¡The function of GABA_A receptors was influenced by PKA phosphorylation and also expressed a heterogeneous character. 1μM forskolin potentiated I_GABA in 77.8%, did not change it in 22.2% of the cells. 1μM H-89 attenuated I_GABA in 69.2% and facilitated it in 30.8% of the cells. Following lμM forskolin pretreatment for 20 minutes, 0.01μM fentanyl inhibited I_GABA to a less extent compared to which without forskolin; on the contrary, fentanyl did not influence I_GABA any more after 1μM H-89 pretreatment, suggesting that the depressant effects of fentanyl on I_GABA was mediated by inhibition of PKA phosphorylation.â‘¢0.3～30μM propofol and 0.03～100μM midazolum facilitated I_GABA dose-dependently with the greatest enhancement at 3μM of each (P<0.05). Following 0.01μM fentanyl pretreatment for 10 minutes, the facilitation of I_GABA by 3μM propofol increased significantly (relative GABA current 2.27 vs. 3.12, P<0.05), while the effects of midazolum did not change, indicating that fentanyl can alter the modulatory response of GABA_A receptors to propofol.Conclusion: Fentanyl can inhibit the function of GABA_A receptors through μ.-opioid receptors and therefore excite the hippocampal pyramidal neurons of neonatal rats. Hippocampus may play a role in the neuroexcitatory effects of opioids. The function of GABA_A receptors was influenced by PKA phosphorylation, enhanced by PKA activation and vise verso. The attenuation of I_GABA by fentanyl was partially blocked by PKA activation and mimicked by PKA inhibition, suggesting that the effects of fentanyl were mediated by PKA inhibition. It is possible that the heterogeneity of GABA responses to fentanyl and PKA is due to the heterogeneous distribution of GABA_A receptors in CA region of rat hippocampus. Propofol and midazolum facilitated I_GABA. Following fentanyl pretreatment, the facilitation of I_GABA by propofol increased, while that of midazolum did not change, implying that fentanyl can alter the modulatory effects of propofol, probably through PKA inhibition. Our study suggests that propofol or midazolum combined with fentanyl may prevent the neuroexcitatory effects of fentanyl, supporting their combination in clinical anesthesia.