GIRK1在颞叶癫痫大鼠海马齿状回中的表达及其意义

目的探讨GIRK1在颞叶癫癎大鼠海马齿状回的表达及其意义.方法112只雄性SD大鼠随机分为实验组(n=70)与对照组(n=42),同时建立海人藻酸(KA)颞叶癫模型.选取KA腹腔注射后3、6、12、24、48 h,7、30 d为研究的时间点.用原位杂交法及免疫组织化学法检测海马齿状回GIRK1 mRNA及蛋白的表达.结果实验组大鼠海马齿状回GIRK1 mRNA表达在致癎后6 h较对照组减少,而在致癎后至7～30 d较对照组增高.结论在颞叶癫癎的不同时期海马齿状回GIRK1表达的变化反映出颞叶癫癎的复杂性.     

碘化N-正丁基氟哌啶醇对豚鼠心房肌细胞乙酰胆碱敏感性钾通道的影响

目的研究碘化N正丁基氟哌啶醇(F2)对豚鼠心房肌细胞乙酰胆碱敏感性钾通道(KACh)的影响,探讨其对KACh的作用机制。方法采用膜片钳全细胞记录方法,测定F2对原代培养的豚鼠心房肌细胞乙酰胆碱敏感性钾电流IK(ACh)的影响。结果细胞外给予F2对豚鼠心房肌细胞IK(ACh)呈可逆性、浓度依赖性的阻断作用。细胞内添入抗水解的GTP类似物GTPγS后,结果同前。细胞内给予50μmol·L-1F2对IK(ACh)无作用。结论F2是豚鼠心房肌细胞KACh的一种快速通道阻断剂,发挥作用部位在细胞膜外侧,作用位点在钾通道本身,与乙酰胆碱受体无关。     

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies “sick sinus” syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Ca_v1.3 Ca²⁺ channels (Ca_v1.3^−/−) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K⁺ channel (I_KACh) could rescue SSS and heart block in Ca_v1.3^−/− mice. We found that genetic inactivation of I_KACh abolished SSS symptoms in Ca_v1.3^−/− mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of I_KACh either in Ca_v1.3^−/− mice or following selective inhibition of Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current in vivo. Ablation of I_KACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from I_KACh suppression achieved by gene therapy or selective pharmacological inhibition.Pacemaker activity of the sinoatrial node (SAN) controls heart rate under physiological conditions. Abnormal generation of SAN automaticity underlies “sick sinus” syndrome (SSS), a pathological condition manifested when heart rate is not sufficient to meet the physiological requirements of the organism (1). Typical hallmarks of SSS include SAN bradycardia, chronotropic incompetence, SAN arrest, and/or exit block (1–3). SSS carries incapacitating symptoms, such as fatigue and syncope (1–3). A significant percentage of patients with SSS present also with tachycardia-bradycardia syndrome (3). SSS can also be associated with atrioventricular (AV) conduction block (heart block) (1–3). Although aging is a known intrinsic cause of SSS (4), this disease appears also in the absence of any associated cardiac pathology and displays a genetic legacy (1, 2). Heart disease or drug intake can induce acquired SSS (2). Symptomatic SSS requires the implantation of an electronic pacemaker. SSS accounts for about half of all pacemaker implantations in the United States (5, 6). The incidence of SSS has been forecasted to increase during the next 50 y, particularly in the elder population (7). Furthermore, it has been estimated that at least half of SSS patients will need to be electronically paced (7). Although pacemakers are continuously ameliorated, they remain costly and require lifelong follow-up. Moreover, the implantation of an electronic pacemaker remains difficult in pediatric patients (8). Development of alternative and complementary pharmacological or molecular therapies for SSS management could improve quality of life and limit the need for implantation of electronic pacemakers.Recently, the genetic bases of some inherited forms of SSS have been elucidated (recently reviewed in 1, 9) with the discovery of mutations in genes encoding for ion channels involved in cardiac automaticity (4, 9, 10). Notably, loss of function of L-type Ca_v1.3 Ca²⁺ channels is central in some inherited forms of SSS. For instance, loss of function in Ca_v1.3-mediated L-type Ca²⁺ (I_Ca,L) current causes the sinoatrial node dysfunction and deafness syndrome (SANDD) (10). Affected individuals with SANDD present with profound deafness, bradycardia, and dysfunction of AV conduction (10). Mutation in ankyrin-B causes SSS by reduced membrane targeting of Ca_v1.3 channels (11). The relevance of Ca_v1.3 channels to SSS is demonstrated also by work on the pathophysiology of congenital heart block, where down-regulation of Ca_v1.3 channels by maternal Abs causes heart block in infants (12). Additionally, recent data show that chronic iron overload induces acquired SSS via a reduction in Ca_v1.3-mediated I_Ca,L (13).In mice and humans, Ca_v1.3 channels are expressed in the SAN, atria, and the AV node but are absent in adult ventricular tissue (14, 15). Ca_v1.3-mediated I_Ca,L plays a major role in the generation of the diastolic depolarization in SAN and AV myocytes, thereby constituting important determinants of heart rate and AV conduction velocity (14, 16). The heart rate of mice lacking Ca_v1.3 channels (Ca_v1.3^−/− mice) fairly recapitulates the hallmarks of SSS and associated symptoms, including bradycardia and tachycardia-bradycardia syndrome (17, 18). In addition, severe AV dysfunction is recorded in Ca_v1.3^−/− mice to variable degrees. Typically, these mice show first- and second-degree AV block (16, 17, 19). Complete AV block with dissociated atrial and ventricular rhythms can also be observed in these animals. The phenotype of Ca_v1.3^−/− mice thus constitutes a unique model for developing new therapeutic strategies against SSS (10).The muscarinic-gated K⁺ channel (I_KACh) is involved in the negative chronotropic effect of the parasympathetic nervous system on heart rate (20, 21). Two subunits of the G-protein activated inwardly rectifying K⁺ channels (GIRK1 and GIRK4) of the GIRK/Kir3 subfamily assemble as heterotetramers to form cardiac I_KACh channels (22). Indeed, both Girk1^−/− and Girk4^−/− mice lack cardiac I_KACh (20, 21, 23). We recently showed that silencing of the hyperpolarization-activated current “funny” (I_f) channel in mice induces a complex arrhythmic profile that can be rescued by concurrent genetic ablation of Girk4 (24). In this study, we tested the effects of genetic ablation and pharmacological inhibition of I_KACh on the Ca_v1.3^−/− mouse model of SSS. We found that Girk4 ablation or pharmacological inhibition of I_KACh rescues SSS and AV dysfunction in Ca_v1.3^−/−. Thus, our study shows that I_KACh targeting may be pursued as a therapeutic strategy for treatment of SSS and heart block.     

Does Acetaldehyde Mediate Ethanol Action in the Central Nervous System?

BACKGROUND: Some of the effects of ethanol in the central nervous system are due to changes in function of ligand-gated ion channels. Production of detectable amounts of acetaldehyde, a primary metabolite of ethanol, has been demonstrated in brain homogenates. The aim of this study was to determine whether central actions that are often attributed to ethanol may actually be mediated by acetaldehyde. METHODS: The effects of acetaldehyde (1-1000 microM) were tested by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing 10 different ligand-gated ion channel receptors [alpha1 glycine; alpha1beta2gamma2Sgamma-aminobutyric acid (GABA)A; rho1 GABAc; 5-hydroxytryptamine-3A; NR1a/NR2A NMDA; GluR1/GluR2 AMPA; GluR6/KA2 kainate; and alpha4beta2, alpha4beta4, and alpha2beta4 nicotinic-acetylcholine] and the G-protein-coupled inward rectifying potassium channel GIRK2. We also investigated the effect of acetaldehyde on the dopamine transporter (DAT), performing dopamine uptake assays in oocytes expressing DAT. RESULTS: Acetaldehyde (1 and 10 microM) significantly enhanced alpha1 glycine receptor-mediated currents. Acetaldehyde did not affect the function of any of the other receptors tested or the potassium currents measured in GIRK2 channels. Moreover, acetaldehyde did not alter the DAT-mediated dopamine uptake. CONCLUSIONS: Our results suggest a potential minor role for acetaldehyde in the glycine receptor-mediated effects of ethanol. Otherwise, acetaldehyde does not modulate function of the neuronal receptors tested in this study, in GIRK channels or DAT, when expressed recombinantly in Xenopus laevis oocytes.     

Antidepressant-like effect of centrally acting non-narcotic antitussive caramiphen in a forced swimming test

Recently, we reported that a centrally acting non-narcotic antitussive (cough suppressant drug), tipepidine produces an antidepressant-like effect in the forced swimming test in rats. Because pharmacological properties of tipepidine apparently differ from those of typical antidepressants developed to date, we speculated that caramiphen, another centrally acting antitussive, has an antidepressant-like effect. That effect of caramiphen was studied in rats using the forced swimming test. Caramiphen at 20 and 40 mg/kg i.p. significantly reduced immobility. At 40 mg/kg i.p., it increased climbing behavior. Even at 40 mg/kg, this drug had no effect on locomotor activity. Results suggest that a centrally acting antitussive possessing inhibition of GIRK channels has an antidepressant-like effect.     

Cell death in weaver mouse cerebellum

Mice with the weaver mutation exhibit an uneven weave to their gait, ataxia, mild locomotor hyperactivity and, occasionally, tonic-clonic seizures. A single amino acid mutation in a G-protein coupled, inwardly rectifying K+ channel, GIRK2, gives rise to the symptoms seen in the weaver mice. Two areas of the brain are primarily affected. Cerebellar granule cell neurons die soon after birth and dopaminergic neurons are severely depleted in the substantia nigra. In this article we review recent studies of wild-type and mutant GIRK channels found in native cells or introduced into expression systems. We also review two models that explain some of the details leading to the neuronal cell death observed in weaver mice.     

Epistatic interaction of CREB1 and KCNJ6 on rumination and negative emotionality

G protein-activated K+ channel 2 (GIRK2) and cAMP-response element binding protein (CREB1) are involved in synaptic plasticity and their genes have been implicated depression and memory processing. Excessive rumination is a core cognitive feature of depression which is also present in remission. High scores on the Ruminative Response Scale (RRS) questionnaire are predictive of relapse and recurrence. Since rumination involves memory, we tested the hypothesis that variation in the genes encoding GIRK2 (KCNJ6) and CREB1 mechanisms would influence RRS scores. GIRK2 and CREB1 polymorphisms were studied in two independent samples (n = 651 and n = 1174) from the general population. Strongly significant interaction between the TT genotype of rs2070995 (located in KCNJ6) and the GG genotype of rs2253206 (located in CREB1) on RRS were found in both samples. These results were validated in an independent third sample (n = 565; individuals with personality disorders) showing significant main effect of the variants mentioned as well as significant interaction on a categorical diagnosis of Cluster C personality disorder (obsessional-compulsive, avoidant and dependent) in which rumination is a prominent feature. Our results suggest that genetic epistasis in post-receptor signaling pathways in memory systems may have relevance for depression and its treatment.     

Ethanol enhances GABAB-mediated inhibitory postsynaptic transmission on rat midbrain dopaminergic neurons by facilitating GIRK currents

It is largely accepted that an activation of the dopaminergic system underlies the recreational and convivial effects of ethanol. However, the mechanisms of action of this drug on the dopaminergic neurons are not fully understood yet. In the present study, we have used intracellular electrophysiological techniques (current and single-electrode voltage-clamp) to investigate the actions of ethanol on the γ-aminobutyric acid (GABA)_B-mediated inhibitory postsynaptic potentials (IPSPs) in rat midbrain dopaminergic neurons. Ethanol (10–200 m m ) augmented, in a concentration-dependent and reversible manner, the amplitude of the GABA_B–IPSP. In addition, the GABA_B agonist baclofen generated G-protein-gated inward rectifying K⁺ channels (GIRK)-related membrane hyperpolarizations/outward currents that were potentiated by ethanol. The potentiating effect of ethanol persisted in tetrodotoxin (TTX)-treated neurons, suggesting a postsynaptic site of action. These effects of ethanol were not changed by manipulating adenyl cyclase, protein kinases and phospholipase C activity, or by chelating intracellular Ca²⁺ with EGTA. Interestingly, the outward current caused by the intracytoplasmatic diffusion of the irreversible G-protein activator GTPγS was transiently enhanced by ethanol. Our observations suggest that the action of ethanol occurs on activated GIRK channels downstream of the GABA_B receptors. These enhancing effects of ethanol on GABA_B-induced synaptic responses could modulate alcohol intake and the altered mental and motor performance of individuals in an acute intoxicative phase.     

The biology of Nociceptin/Orphanin FQ (N/OFQ) related to obesity,stress, anxiety,mood, and drug dependence

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995. The generation of specific agonists, antagonists and receptor deficient mice and rats has enabled progress in elucidating the biological functions of N/OFQ. Additionally, radio-imaging technologies have been advanced for investigation of this system in animals and humans. Together with traditional neurobehavioral techniques, these tools have been utilized to identify the biological significance of the N/OFQ system and its interacting partners. The present review focuses on the role of N/OFQ in the regulation of feeding, body weight homeostasis, stress, the stress-related psychiatric disorders of depression and anxiety, and in drug and alcohol dependence. Critical evaluation of the current scientific preclinical literature suggests that small molecule modulators of nociceptin opioid peptide receptors (NOP) might be useful in the treatment of diseases related to these biological functions. In particular, the literature data suggest that antagonism of NOP receptors will produce anti-obesity and antidepressant activities in humans. However, there are also contradictory data discussed. The current literature on the role of N/OFQ in anxiety and addiction, on the other hand points primarily to a role of agonist modulation being potentially therapeutic. Some drug-like molecules that function either as agonists or antagonists of NOP receptors have been optimized for human clinical study to test some of these hypotheses. The discovery of PET ligands for NOP receptors, combined with the pharmacological tools and burgeoning preclinical data set discussed here bodes well for a rapid advancement of clinical understanding and potential therapeutic benefit.