Two SUR1-specific histidine residues mandatory for zinc-induced activation of the rat KATP channel

Zinc at micromolar concentrations hyperpolarizes rat pancreatic beta-cells and brain nerve terminals by activating ATP-sensitive potassium channels (KATP). The molecular determinants of this effect were analyzed using insulinoma cell lines and cells transfected with either wild type or mutated KATP subunits. Zinc activated KATP in cells co-expressing rat Kir6.2 and SUR1 subunits, as in insulinoma cell lines. In contrast, zinc exerted an inhibitory action on SUR2A-containing cells. Therefore, SUR1 expression is required for the activating action of zinc, which also depended on extracellular pH and was blocked by diethyl pyrocarbonate, suggesting histidine involvement. The five SUR1-specific extracellular histidine residues were submitted to site-directed mutagenesis. Of them, two histidines (His-326 and His-332) were found to be critical for the activation of KATP by zinc, as confirmed by the double mutation H326A/H332A. In conclusion, zinc activates KATP by binding itself to extracellular His-326 and His-332 of the SUR1 subunit. Thereby zinc could exert a negative control on cell excitability and secretion process of pancreatic beta-and alpha-cells. In fact, we have recently shown that such a mechanism occurs in hippocampal mossy fibers, a brain region characterized, like the pancreas, by an important accumulation of zinc and a high density of SUR1-containing KATP.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

成年大鼠海马CA1区锥体细胞KATP通道的特性

为了解成年大鼠海马CA1区锥体细胞KATP通道的特性,实验采用膜片钳技术的内面向外式记录法,在急性分离的CA1区锥体神经元上,研究了可被胞浆侧ATP所抑制的钾离子单通道的特性,当细胞膜内外两侧的K^ 浓度均为140mmol/L时,通道的电导为63pS,翻转电位为1．71mV,通道呈弱向内向整流性,在负钳制电位时,通道开放时常被短时的关闭所打断,而在正钳制电位时,这种短时程的关闭状态明显少于负钳制电位时,但通道开放概率未见明显的电压依赖性,ATP对通道活动的抑制作用呈浓度依赖性,抑制通道活动50％的ATP浓度为0．1mmol/L.KATP通道的特异性阻断剂tolbutamide(甲糖宁,1mmol/L)可完全阻断通道的活动,而KATP通道开放剂diazoxide(二氮嗪,1mmol/L）则不增强通道的活动。     

Activation of mitochondrial ATP-sensitive potassium channels increases cell viability against rotenone-induced cell death

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.     

Regulation of mitochondrial KATP channel by redox agents

The ATP-dependent K+ channel (KATP) was purified from the inner mitochondrial membrane and reconstituted into lipid bilayer membranes. KATP activity was inhibited by high concentrations of ATP and ADP, but activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethylbenzoate (DEB) acted as a KATP opener: at micromolar concentrations, it reversed inhibition by ATP and ADP and it also prevented KATP rundown. Pelargonidine, extracted from flowers of Pelargonium, reduced spontaneous activity of KATP channels and diminished their potentiation by DEB. Their opposite action on KATP corresponded with their opposite redox properties in reactions with free radicals: DEB behaved as an electron donor, whereas pelargonidine acted as an electron acceptor. We hypothesize that thiol groups on mitoKATP are targets for redox-active ligans.     

Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K+ channels in rat hearts

Nitric oxide (NO) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries. The present work was performed to study better the NO-cGMP-protein kinase G (PKG) signaling pathway in the activation of both sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels during anoxic preconditioning (APC) and final influence on reducing anoxia-reperfusion (A/R)-induced cardiac damage in rat hearts. The upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection were investigated. The involvement of both inducible and endothelial NO synthases (iNOS and eNOS) in the progression of this signaling pathway was followed. Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release, DNA strand breaks, and malondialdehyde formation as indexes of cell injury and lipid peroxidation, respectively, were investigated. The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial KATP channels opener diazoxide (100 microM), nonspecific mitochondrial KATP channels opener pinacidil (50 microM), S-nitroso-N-acetylpenicillamine (SNAP, 300 microM), or beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclicmonophosphorothioate, Sp-isomer (10 microM) before the A/R period. Preconditioning with SNAP significantly reduced the DNA damage. The effect was blocked by glibenclamide (50 microM), 5-hydroxydecanoate (100 microM), NG-nitro-L-arginine methyl ester (200 microM), and beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (1 microM). The results suggest iNOS, rather than eNOS, as the major contributing NO synthase during APC treatment. Moreover, the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection during APC treatment.     

Opening of Ca2+-activated K+ channels triggers early and delayed preconditioning against I/R injury independent of NOS in mice

Opening of Ca2+-activated K+ (KCa) channels has been shown to confer early cardioprotection. It is unknown whether the opening of these channels also induces delayed cardioprotection. In addition, we determined the involvement of nitric oxide synthases (NOSs), which have been implicated in cardioprotection induced by opening of mitochondrial ATP-sensitive K+ (KATP) channels. Adult male ICR mice were pretreated with the KCa-channel opener NS-1619 either 10 min or 24 h before 30 min of global ischemia and 60 min of reperfusion (I/R) in Langendorff mode. Infusion of NS-1619 (10 microM) for 10 min before I/R led to smaller infarct sizes as compared with the vehicle (DMSO)-treated group (P <0.05). This infarct-limiting effect of NS-1619 was associated with improvement in ventricular functional recovery after I/R. The NS-1619-induced protection was abolished by coadministration with the KCa-channel blocker paxilline (1 microM). Similarly, pretreatment with NS-1619 (1 mg/kg ip) induced delayed protection 24 h later (P <0.05). Interestingly, the NS-1619-induced late protection was not blocked by the NOS inhibitor Nomega-nitro-L-arginine methyl ester (15 mg/kg ip). Unlike diazoxide (the opener of mitochondrial KATP channels), NS-1619 did not increase the expression of inducible or endothelial NOS. Western blot analysis demonstrated the existence of alpha- and beta-subunits of KCa channels in mouse heart tissue. We conclude that opening of KCa channels leads to both early and delayed preconditioning effects through a mechanism that is independent of nitric oxide.     

内皮素和ATP敏感性钾通道介导缺氧所致家兔窦房结起搏细胞的电生理效应

用细胞内微电极技术研究了ＡＴＰ－敏感性钾（Ｋ_（ＡＴＰ））通道和内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）在缺氧所致窦房结起搏细胞负性频率中的作用,主要结果如下：（１）缺氧引起窦房结起搏细胞的ＲＰＦ降低和ＡＰＤ缩短,这一效应随时间延长而加重。（２）Ｋ_（ＡＴＰ）通道开放剂ｃｒｏｍａｋａｌｉｍ浓度依赖性地对窦房结起搏细胞有负性频率作用,且明显缩短ＡＰＤ_（５０）。该通道的阻断剂格列苯脲能部分阻断缺氧对起搏细胞的上述效应,表明缺氧效应中有Ｋ_（ＡＴＰ）通道的参与。（３）ＥＴ－１可显著加重缺氧所致的ＲＰＦ降低,使起搏细胞停跳时间前移;而以ＥＴ_Ａ受体阻断剂ＢＱ－１２３预处理窦房结标本后,则能有效地缓解缺氧对起搏细胞的效应,提示内源性ＥＴ－１的释放在缺氧效应中的作用。上述结果表明,缺氧所致起搏细胞的负性频率作用和ＡＰＤ缩短,与Ｋ_（ＡＴＰ）通道的激活和内源性ＥＴ－１的释放有关。     

ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.     

Effect of ATP sensitive potassium channel modifiers on antinociceptive effect of metoclopramide

Metoclopramide, a prokinetic drug, has been documented to produce antinociceptive response in animal models through opioid pathways. Morphine has been shown to act through ATP sensitive potassium channels (KATP) to produce antinociceptive response. However, such a possibility has not been examined for metoclopramide. The present study investigated this using pharmacological tools. Acetic acid induced abdominal constriction assay procedure was utilized to assess antinociception. The results confirmed that metoclopramide has antinociceptive response. Glibenclamide, a KATP channel blocker, pretreatment antagonized this response. Where as, in minoxidil pretreated animals, metoclopramide elicited an enhanced antinociceptive response. Glibenclamide and minoxidil, which are known KATP channel blocker and opener respectively, interfered with metoclopramide antinociception. These finding are suggestive of a role for KATP channels in metoclopramide antinociception in mice.     

12-Lipoxygenase Products Attenuate the Glutamate Release and Ca2+ Accumulation Evoked by Depolarization of Hippocampal Mossy Fiber Nerve Endings

Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium-dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12-lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5- or 15-lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1-10 microM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12-lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.     

Response of hippocampal mossy fiber zinc to excessive glutamate release

The response of hippocampal mossy fiber zinc to excessive glutamate release was examined to understand the role of the zinc in excessive excitation in the hippocampus. Extracellular zinc and glutamate concentrations during excessive stimulation with high K(+) were compared between the hippocampal CA3 and CA1 by the in vivo microdialysis. Zinc concentration in the CA3 was more increased than that in the CA1, while glutamate concentration in the CA3 was less increased than that in the CA1. It is likely that more increase in extracellular zinc is linked with less increase in extracellular glutamate in the CA3. To see zinc action in mossy fiber synapses during excessive excitation, furthermore, 1mM glutamate was regionally delivered to the stratum lucidum in the presence of zinc or CaEDTA, a membrane-impermeable zinc chelator, and intracellular calcium signal was measured in the CA3 pyramidal cell layer. The persistent increase in calcium signal during stimulation with glutamate was significantly attenuated in the presence of 100 microM zinc, while significantly enhanced in the presence of 1mM CaEDTA. These results suggest that zinc released from mossy fibers attenuates the increase in intracellular calcium signal in mossy fiber synapses and postsynaptic CA3 neurons after excessive inputs to dentate granular cells.     

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.     

Do modulators of the mitochondrial K(ATP) channel change the function of mitochondria in situ?

Pharmacological opening of mitochondrial cardiac ATP-sensitive potassium (K(ATP)) channels has the chance to be a promising but still controversial cardioprotective mechanism. Physiological roles of mitochondrial K(ATP) channels in the myocardium remain unclear. We studied the effects of diazoxide, a specific opener of these channels, on the function of rat mitochondria in situ in saponin-permeabilized fibers using an ionic medium that mimics the cytosol. In the presence of NADH-producing substrates (malate + glutamate), neither 100 microm diazoxide nor 100 microm glibenclamide (a K(ATP) channel blocker) changed the mitochondrial respiration in the absence or presence of ADP. Because the K(ATP) channel function could be modified by changes in adenine nucleotide concentrations near the mitochondria, we studied the effects of diazoxide and glibenclamide on the functional activity of mitochondrial kinases. Both diazoxide and glibenclamide did not change the in situ ADP sensitivity in the presence or absence of creatine (apparent K(m) values for ADP were, respectively, 59 +/- 9 and 379 +/- 45 microm). Similarly, stimulation of the mitochondrial respiration with AMP in the presence of ATP due to adenylate kinase activity was not affected by the modulators of K(ATP) channels. However, when succinate was used as substrate, diazoxide significantly inhibited basal respiration by 22% and maximal respiration by 24%. Thus, at a cardioprotective dose, the main functional effect of diazoxide depends on respiratory substrates and seems not to be related to K(ATP) channel activity.     

Modulation of the c-Jun N-terminal kinase activity in the embryonic heart in response to anoxia-reoxygenation: involvement of the Ca2+ and mitoKATP channels

Whether the response of the fetal heart to ischemia-reperfusion is associated with activation of the c-Jun N-terminal kinase (JNK) pathway is not known. In contrast, involvement of the sarcolemmal L-type Ca2+ channel (LCC) and the mitochondrial KATP (mitoKATP) channel has been established. This work aimed at investigating the profile of JNK activity during anoxia-reoxygenation and its modulation by LCC and mitoK(ATP) channel. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (60 min). Using the kinase assay method, the profile of JNK activity in the ventricle was determined every 10 min throughout anoxia-reoxygenation. Effects on JNK activity of the LCC blocker verapamil (10 nM), the mitoK(ATP) channel opener diazoxide (50 microM) and the blocker 5-hydroxydecanoate (5-HD, 500 microM), the mitochondrial Ca2+ uniporter (MCU) inhibitor Ru360 (10 microM), and the antioxidant N-(2-mercaptopropionyl) glycine (MPG, 1 mM) were determined. In untreated hearts, JNK activity was increased by 40% during anoxia and peaked fivefold relative to basal level after 30-40 min reoxygenation. This peak value was reduced by half by diazoxide and was tripled by 5-HD. Furthermore, the 5-HD-mediated stimulation of JNK activity during reoxygenation was abolished by diazoxide, verapamil or Ru360. MPG had no effect on JNK activity, whatever the conditions. None of the tested pharmacological agents altered JNK activity under basal normoxic conditions. Thus, in the embryonic heart, JNK activity exhibits a characteristic pattern during anoxia and reoxygenation and the respective open-state of LCC, MCU and mitoKATP channel can be a major determinant of JNK activity in a ROS-independent manner.     

Syntaxin-1A binds the nucleotide-binding folds of sulphonylurea receptor 1 to regulate the KATP channel

ATP-sensitive potassium (KATP) channels in neuron and neuroendocrine cells consist of a pore-forming Kir6.2 and regulatory sulfonylurea receptor (SUR1) subunits, which are regulated by ATP and ADP. SNARE protein syntaxin 1A (Syn-1A) is known to mediate exocytic fusion, and more recently, to also bind and modulate membrane-repolarizing voltage-gated K+ channels. Here we show that Syn-1A acts as an endogenous regulator of KATP channels capable of closing these channels when cytosolic ATP concentrations were lowered. Botulinum neurotoxin C1 cleavage of endogenous Syn-1A in insulinoma HIT-T15 cells resulted in the increase in KATP currents, which could be subsequently inhibited by recombinant Syn-1A. Whereas Syn-1A binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1, the functional inhibition of KATP channels in rat islet beta-cells by Syn-1A seems to be mediated primarily by its interactions with NBF-1. These inhibitory actions of Syn-1A can be reversed by physiologic concentrations of ADP and by diazoxide. Syn-1A therefore acts to fine-tune the regulation of KATP channels during dynamic changes in cytosolic ATP and ADP concentrations. These actions of Syn-1A on KATP channels contribute to the role of Syn-1A in coordinating the sequence of ionic and exocytic events leading to secretion.     

Modulation of neuronal voltage-gated calcium channels by farnesol.

The modulation of presynaptic voltage-dependent calcium channels by classical second messenger molecules such as protein kinase C and G protein betagamma subunits is well established and considered a key factor for the regulation of neurotransmitter release. However, little is known of other endogenous mechanisms that control the activity of these channels. Here, we demonstrate a unique modulation of N-type calcium channels by farnesol, a dephosphorylated intermediate of the mammalian mevalonate pathway. At micromolar concentrations, farnesol acts as a relatively non-discriminatory rapid open channel blocker of all types of high voltage-activated calcium channels, with a mild specificity for L-type channels. However, at 250 nM, farnesol induces an N-type channel-specific hyperpolarizing shift in channel availability that results in approximately 50% inhibition at a typical neuronal resting potential. Additional experiments demonstrated the presence of farnesol in the brain (rodents and humans) at physiologically relevant concentrations (100-800 pmol/g (wet weight)). Altogether, our results indicate that farnesol is a selective, high affinity inhibitor of N-type Ca(2+) channels and raise the possibility that endogenous farnesol and the mevalonate pathway are implicated in neurotransmitter release through regulation of presynaptic voltage-gated Ca(2+) channels.     

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.     

Cell-type specific depression of neuronal excitability in rat hippocampus by activation of ATP-sensitive potassium channels

The contribution of ATP-sensitive potassium (K(ATP)) channels to neuronal excitability was studied in different types of pyramidal cells and interneurones in hippocampal slices prepared from 9- to 15-day-old rats. The presence of functional K(ATP) channels in the neurones was detected through the sensitivity of whole-cell currents to diazoxide, a K(ATP) channel opener, and to tolbutamide, a K(ATP) channel inhibitor. The percentages of neurones with K(ATP) channels increase in the sequence: CA1 pyramidal cells (37%)相似文献