Altered Expression Pattern of Acid-Sensing Ion Channel Isoforms in Piriform Cortex After Seizures
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Piriform cortex
Acid-sensing ion channel
Amiloride
Pilocarpine
Acid-sensing ion channel
Amiloride
Capsazepine
Nociceptor
Capsaicin
Epithelial sodium channel
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Pilocarpine-induced seizures in rats provide a widely animal model of temporal lobe epilepsy. Some evidences reported in the literature suggest that at least 1 h of status epilepticus (SE) is required to produce subsequent chronic phase, due to the SE-related acute neuronal damage. However, recent data seems to indicate that neuro-inflammation plays a crucial role in epileptogenesis, modulating secondarily a neuronal insult. For this reason, we decided to test the following hypotheses: a) whether pilocarpine-injected rats that did not develop SE can exhibit long-term chronic spontaneous recurrent seizures (SRS) and b) whether acute neurodegeneration is mandatory to obtain chronic epilepsy. Therefore, we compared animals injected with the same dose of pilocarpine that developed or did not SE, and saline treated rats. We used telemetric acquisition of EEG as long-term monitoring system to evaluate the occurrence of seizures in non-SE pilocarpineinjected animals. Furthermore, histology and MRI analysis were applied in order to detect neuronal injury and neuropathological signs. Our observations indicate that non-SE rats exhibit SRS almost 8 (+/22) months after pilocarpine-injection, independently to the absence of initial acute neuronal injury. This is the first time reported that pilocarpine injected rats without developing SE, can experience SRS after a long latency period resembling human pathology. Thus, we strongly emphasize the important meaning of including these animals to model human epileptogenesis in pilocarpine induced epilepsy.
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Acid-Sensing Ion Channels (ASICs) are proton-gated sodium-selective cation channels that have emerged as metabolic and pain sensors in peripheral sensory neurons and contribute to neurotransmission in the CNS. These channels and their related degenerin/epithelial sodium channel (DEG/ENaC) family are often characterized by their sensitivity to amiloride inhibition. However, amiloride can also cause paradoxical potentiation of ASIC currents under certain conditions. Here we characterized and investigated the determinants of paradoxical potentiation by amiloride on ASIC3 channels. While inhibiting currents induced by acidic pH, amiloride potentiated sustained currents at neutral pH activation. These effects were accompanied by alterations in gating properties including (1) an alkaline shift of pH-dependent activation, (2) inhibition of pH-dependent steady-state desensitization (SSD), (3) prolongation of desensitization kinetics, and (4) speeding of recovery from desensitization. Interestingly, extracellular Ca2+ was required for paradoxical potentiation and it diminishes the amiloride-induced inhibition of SSD. Site-directed mutagenesis within the extracellular non-proton ligand-sensing domain (E79A, E423A) demonstrated that these residues were critical in mediating the amiloride-induced inhibition of SSD. However, disruption of the purported amiloride binding site (G445C) within the channel pore blunted both the inhibition and potentiation of amiloride. Together, our results suggest that the myriad of modulatory and blocking effects of amiloride are the result of a complex competitive interaction between amiloride, Ca2+, and protons at probably more than one site in the channel.
Amiloride
Acid-sensing ion channel
Epithelial sodium channel
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Amiloride
Acid-sensing ion channel
Epithelial sodium channel
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Piriform cortex
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ABSTRACT Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K + -sparing diuretic amiloride is a moderate non-specific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch-clamp protocol and identified 6-iodoamiloride as a more potent ASIC1 inhibitor. Follow-up IC 50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 97 (hASIC1 97 IC 50 88 nM cf. amiloride 11 IC 50 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat small diameter dorsal root ganglion neurons (rDRG single-concentration 97 IC 50 230 nM cf. 11 IC 50 2.7 μM). 6-iodoamiloride represents the amiloride analogue of choice for studying the effects of ASIC inhibition on cell physiology.
Amiloride
Acid-sensing ion channel
Dorsal root ganglion
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Pilocarpine
Subthreshold conduction
Pentylenetetrazol
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Pilocarpine
Frontal cortex
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Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 μM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.
Amiloride
Acid-sensing ion channel
Dorsal root ganglion
IC50
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Summary Background ASIC 1a, the predominant acid‐sensing ion channels ( ASIC s), is implicated in neurological disorders including stroke, traumatic spinal cord injury, and ALS . Potent ASIC 1a inhibitors should have promising therapeutic potential for ASIC 1a‐related diseases. Aims We examined the inhibitory effects of a number of amiloride analogs on ASIC 1a currents, aimed at understanding the structure–activity relationship and identifying potent ASIC 1a inhibitors for stroke intervention. Methods Whole‐cell patch‐clamp techniques and a mouse model of middle cerebral artery occlusion ( MCAO )‐induced focal ischemia were used. Surflex‐Dock was used to dock the analogs into the pocket with default parameters. Results Amiloride and its analogs inhibit ASIC 1a currents expressed in Chinese hamster ovary cells with a potency rank order of benzamil > phenamil > 5‐( N , N ‐dimethyl)amiloride ( DMA ) > amiloride > 5‐( N , N ‐hexamethylene)amiloride ( HMA ) ≥ 5‐( N ‐methyl‐ N ‐isopropyl)amiloride ( MIA ) > 5‐( N ‐ethyl‐ N ‐isopropyl)amiloride ( EIPA ). In addition, amiloride and its analogs inhibit ASIC currents in cortical neurons with the same potency rank order. In mice, benzamil and EIPA decreased MCAO ‐induced infarct volume. Similar to its effect on the ASIC current, benzamil showed a much higher potency than EIPA . Conclusion Addition of a benzyl group to the terminal guanidinyl group resulted in enhanced inhibitory activity on ASIC 1a. On the other hand, the bulky groups added to the 5‐amino residues slightly decreased the activity. Among the tested amiloride analogs, benzamil is the most potent ASIC 1a inhibitor.
Amiloride
Acid-sensing ion channel
Epithelial sodium channel
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