Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

Elabela relaxes rat pulmonary artery and trachea via BKCa, KV, and KATP channels.

OBJECTIVE: Elabela is a newly discovered peptide hormone. This study aimed to determine the functional effects and mechanisms of action of elabela in rat pulmonary artery and trachea. MATERIALS AND METHODS: Vascular rings isolated from the pulmonary arteries of male Wistar Albino rats were placed in chambers in the isolated tissue bath system. The resting tension was set to 1 g. After the equilibration period, the pulmonary artery rings were contracted with 10-6 M phenylephrine. Once a stable contraction was achieved, elabela was applied cumulatively (10-10-10-6 M) to the vascular rings. To determine the vasoactive effect mechanisms of elabela, the specified experimental protocol was repeated after the incubation of signaling pathway inhibitors and potassium channel blockers. The effect and mechanisms of action of elabela on tracheal smooth muscle were also determined by a similar protocol. RESULTS: Elabela exhibited a concentration-dependent relaxation in the precontracted rat pulmonary artery rings (p < .001). Maximal relaxation level was 83% (pEC50: 7.947 CI95(7.824-8.069)). Removal of the endothelium, indomethacin incubation, and dideoxyadenosine incubation significantly decreased the vasorelaxant effect levels of elabela (p < .001). Elabela-induced vasorelaxation levels were significantly reduced after iberiotoxin, glyburide, and 4-Aminopyridine administrations (p < .001). L-NAME, methylene blue, apamin, TRAM-34, anandamide, and BaCl2 administrations did not cause a significant change in the vasorelaxant effect level of elabela (p = 1.000). Elabela showed a relaxing effect on precontracted tracheal rings (p < .001). Maximal relaxation level was 73% (pEC50: 6.978 CI95(6.791-7.153)). The relaxant effect of elabela on tracheal smooth muscle was decreased significantly after indomethacin, dideoxyadenosine, iberiotoxin, glyburide, and 4-Aminopyridine incubations (p < .001). CONCLUSIONS: Elabela exerted a prominent relaxant effect in the rat pulmonary artery and trachea. Intact endothelium, prostaglandins, cAMP signaling pathway, and potassium channels (BKCa, KV, and KATP channels) are involved in the vasorelaxant effect of elabela. Prostaglandins, cAMP signaling pathway, BKCa channels, KV channels, and KATP channels also contribute to elabela-induced tracheal smooth muscle relaxant effect.

Effects of 4-Aminopyridine on Combined Nerve and Muscle Injury and Bone Loss.

PURPOSE: Musculoskeletal injuries are common, and peripheral nerve injury (PNI) causes significant muscle and bone loss within weeks. After PNI, 4-aminopyridine (4-AP) improves functional recovery and muscle atrophy. However, it is unknown whether 4-AP has any effect on isolated traumatic muscle injury and PNI-induced bone loss. METHODS: A standardized crush injury was performed on the sciatic nerve and muscles in mice, and the mice were assigned to receive normal saline or 4-AP treatment daily for 21 days. The postinjury motor and sensory function recovery was assessed, injured muscles were processed for histomorphometry, and the tibial bone was scanned for bone density. RESULTS: 4-Aminopyridine significantly accelerated the postinjury motor and sensory function recovery, improved muscle histomorphometry, increased muscle satellite cell numbers, and shifted muscle fiber types after combined nerve and muscle injury. Importantly, the 4-AP treatment significantly reduced PNI-induced bone loss. In contrast, in the case of isolated muscle injury, 4-AP had no effect on functional recovery and bone density, but it improved muscle-specific histomorphometry to a limited extent. CONCLUSIONS: These findings demonstrate the potential beneficial effects of 4-AP on the recovery of muscle morphology and bone density after combined muscle and nerve injury. CLINICAL RELEVANCE: Nerve injuries frequently involve muscle and result in rapid muscle and bone atrophy. In this scenario, 4-AP, in addition to accelerating nerve functional recovery, might work as an adjunctive agent to improve the recovery of injured muscle and attenuate PNI-induced bone loss.

Pharmacological evidence that potassium channels mediate hydrogen sulfide-induced inhibition of the vasopressor sympathetic outflow in pithed rats.

Hydrogen sulfide (H2S) is a gasotransmitter that modulates neurotransmission. Indeed, it has been recently demonstrated that H2S inhibits the sympathetic outflow in male rats, although the mechanisms remain elusive. Thus, this study evaluated the role of potassium channels on NaHS-induced sympathoinhibition. For this purpose, male and female Wistar rats were anesthetized, pithed, and cannulated. After that, animals received selective electrical stimulation of the vasopressor sympathetic outflow (T7-T9). Prior to 310 µg/kg·min NaHS i.v. continuous infusion animals received: (1) bidistilled water (tetraethylammonium, TEA; 4-aminopyridine, 4-AP; and barium chloride, BaCl2; vehicle; 1 ml/kg); (2) TEA (non-selective K+ channels blocker; 16.5 mg/kg); (3) 4-AP (non-selective voltage-dependent K+ channels blocker; 5 mg/kg); (4) BaCl2 (inward rectifier K+ channels blocker; 65 µg/kg); (5) DMF 5%, glucose 10% and NaOH 0.1 N (glibenclamide vehicle; 1 ml/kg); (6) glibenclamide (ATP-dependent K+ channels blocker; 10 mg/kg); (7) DMSO 4% (paxilline vehicle; 1 ml/kg); and (8) paxilline (large-conductance voltage- and Ca2+-activated K+ channel blocker; 90 µg/kg). The NaHS-induced sympathoinhibition was: (1) equally observed in male and female rats; (2) unaffected by vehicles; (3) reversed by the potassium channel blockers. Taken together, our results suggest that NaHS-induced sympathoinhibition does not depend on sex and it is mediated by the activation of several potassium channels.

Lappaconitine inhibits glutamate release from rat cerebrocortical nerve terminals by suppressing Ca2+ influx and protein kinase A cascade.

The inhibition of the excessive release of glutamate in the brain has emerged as a promising new option for developing therapeutic strategies for neurodegenerative disorders. This study investigated the effect and mechanism of lappaconitine, a diterpenoid alkaloid found in species of Aconitum, on glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Here, we report that in the rat cortical synaptosomal preparation, lappaconitine reduced the K+ channel blocker 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate. The inhibitory effect of lappaconitine on the evoked glutamate release was blocked by the vesicular transporter inhibitor bafilomycin A1 and calcium-chelating agent ethylene glycol tetraacetic acid (EGTA), but was unaffected by exposure to the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate (dl-TBOA). The depolarization-induced elevation of cytosolic calcium concentration ([Ca2+]c) was inhibited by lappaconitine, while the 4-AP-mediated depolarization of the synaptosomal membrane potential was not affected. The inhibition of glutamate release by lappaconitine was markedly decreased in synaptosomes pretreated with the Cav2.3 (R-type) channel blocker SNX-482 or the protein kinase A inhibitor H89. Nevertheless, the lappaconitine-mediated inhibition of glutamate release was not abolished by the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Lappaconitine also significantly decreased the 4-AP-induced phosphorylation of PKA and SNAP-25, a presynaptic substrate for PKA. Our data suggest that lappaconitine reduces Ca2+ influx through R-type Ca2+ channels, subsequently reducing the protein kinase A cascade to inhibit the evoked glutamate release from rat cerebral cortex nerve terminals.

The role of potassium channels in the proliferation and migration of endometrial adenocarcinoma HEC1-A cells.

BACKGROUND: Endometrial cancer is the most common gynecological cancer in developed countries. Potassium channels, which have many types, are suggested to play a major role in cancer progression. However, their role in endometrial cancer has not been fully investigated. We aimed to demonstrate whether the ATP-sensitive potassium channel blocker glibenclamide, voltage-sensitive potassium channel blocker 4-aminopyridine, non-selective (voltage-sensitive and calcium-activated) potassium channels blocker tetraethylammonium and potassium chloride (KCl) have any effect on the proliferation and migration of HEC1-A cells. METHODS AND RESULTS: Proliferation and migration were evaluated by real-time cell analysis (xCELLigence system) and wound healing assays, respectively. Proliferation was reduced by glibenclamide (0.1 and 0.2 mM, P < 0.05 and P < 0.01, respectively), 4-aminopyridine (10 and 20 mM, P < 0.001) and tetraethylammonium (10 and 20 mM, P < 0.01 and P < 0.001, respectively). However, KCl did not change the proliferation. Migration was reduced by glibenclamide (0.01, 0.1 and 0.2 mM, P < 0.001, P < 0.001 and P < 0.01, respectively) and 4-aminopyridine (10 and 20 mM, P < 0.05 and P < 0.01, respectively). Tetraethylammonium did not change migration. However, KCl reduced it (10, 25 and 50 mM, P < 0.05, P < 0.01 and P < 0.01, respectively). Both proliferation and migration were reduced by glibenclamide and 4-aminopyridine. However, tetraethylammonium only reduced proliferation and KCl only reduced migration. CONCLUSIONS: Potassium channels have an important role in HEC1-A cell proliferation and migration and potassium channel blockers needs to be further investigated for their therapeutic effect in endometrial cancer.

Reversal effects of atipamezole, flumazenil, and 4-aminopyridine on bradycardia and increases in blood pressures induced by medetomidine, midazolam, and ketamine in isoflurane-anesthetized cats.

OBJECTIVE: To investigate the effects of a fixed dose of atipamezole (AT), flumazenil (FL), and 4-aminopyridine (AP), both alone and in combination, on changes in arterial blood pressure and heart rate induced by medetomidine (ME), midazolam (MI), and ketamine (KE) under isoflurane anesthesia with controlled ventilation in healthy cats. DESIGN: Prospective experimental study. SETTING: University animal research facility. ANIMALS: Healthy adult mixed-breed cats were used for 8 investigation groups (6 cats per group), with ≥2 weeks between interventions. INTERVENTIONS: Cats were anesthetized with an end-tidal isoflurane concentration of 2% under controlled ventilation. A catheter was inserted into the right or left femoral artery for arterial pressure monitoring and blood gas sampling, and ECG electrodes were placed. Upon completed preparations, cats were administered a mixture of ME (0.05 mg/kg) and MI (0.5 mg/kg), followed 10 minutes later by intramuscular KE (10 mg/kg). Twenty minutes after KE injection, the cats received IV injection with either a physiological saline solution at 0.1 mL/kg (control), or 1 of 7 variations of experimental drugs, alone or in combination: AT (0.2 mg/kg), FL (0.1 mg/kg), AP (0.5 mg/kg), AT+FL, FL+AP, AT+AP, and AT+FL+AP. Arterial blood pressure and heart rate were continuously measured over 120 minutes after administration of potential antagonists. MEASUREMENTS AND MAIN RESULTS: ME+MI+KE induced an increase in blood pressure and bradycardia. Potential antagonists alone or in combination did not significantly alter the bradycardia. FL, AP alone, and FL+AP did not significantly alter the changes in blood pressures induced by ME+MI+KE. Meanwhile, administration of AT alone or in combination reversed the increase in blood pressure induced by ME+MI+KE but transiently caused excessive hypotension. CONCLUSION: These results revealed that AT alone or in combination is effective for antagonizing hypertension induced by ME+MI+KE; however, attention should be paid to temporary hypotension in cats anesthetized with isoflurane.

Rosmarinic Acid, a Bioactive Phenolic Compound, Inhibits Glutamate Release from Rat Cerebrocortical Synaptosomes through GABAA Receptor Activation.

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.

Roles of voltage­gated potassium channels in the maintenance of pancreatic cancer stem cells.

The targeting of membrane proteins that are activated in cancer stem cells (CSCs) represents one of the key recent strategies in cancer therapy. The present study analyzed ion channel expression profiles and functions in pancreatic CSCs (PCSCs). Cells strongly expressing aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were isolated from the human pancreatic PK59 cell line using fluorescence­activated cell sorting, and PCSCs were identified based on tumorsphere formation. Microarray analysis was performed to investigate the gene expression profiles in PCSCs. ALDH1A1 messenger RNA levels were higher in PCSCs compared with non­PCSCs. PCSCs were resistant to 5­fluorouracil and capable of redifferentiation. The results of the microarray analysis revealed that gene expression related to ion channels, including voltage­gated potassium channels (Kv), was upregulated in PCSCs compared with non­PCSCs. 4­Aminopyridine (4­AP), a potent Kv inhibitor, exhibited greater cytotoxicity in PCSCs compared with non­PCSCs. In a xenograft model in nude mice, tumor volumes were significantly lower in mice inoculated with PK59 cells pre­treated with 4­AP compared with those in mice injected with non­treated cells. The present results identified a role of Kv in the persistence of PCSCs and suggested that the Kv inhibitor 4­AP may have potential as a therapeutic agent for pancreatic carcinoma.

The effects of 4-aminopyridine and methylprednisolone on recovery of the facial nerve crush injury.

OBJECTIVE: 4-Aminopyridine (4-AP) is a potassium channel blocker that enhances nerve excitability. In this study, rat models that have facial nerve crush injury (FNCI) were grouped and treated with methylprednisolone (MP), 4-AP, and a combination of these two drugs. Electrophysiologic and histopathologic outcomes of these groups will be compared with a control group. MATERIALS AND METHODS: Thirty healthy male Wistar rats (mean weight of 265 g) were used in this study. The rats were randomly divided into five groups with six subjects in each: Group 1 (sham group), Group 2 (control group), Group 3 (MP group), Group 4 (4-aminopyridine group), and Group 5 (4-AP + MP group). All groups except the sham group underwent crush injury to the right facial nerve. Electrophysiologic and histologic recovery was recorded three weeks postoperatively. RESULTS: The 4-AP group and the combined group had a more significant recovery at Nerve Excitability Thresholds (NET) at the end of three weeks. The methylprednisolone group and the control group had a minimal recovery of NET. Histologically, when compared with the control group, the combined group was the only group that had significant recovery at all three of axonal degeneration, axon diameter, and myelin thickness. CONCLUSION: In this experimental study, we demonstrated that a combination treatment of 4-AP and MP is more effective in the recovery of peripheric FNCI than in the no-treatment control group and in the 4-AP- or MP-alone groups. Moreover, our results suggested that 4-AP can be a potent alternative to MP in the treatment of the FNCI. LEVEL OF EVIDENCE: N/A.

Bursting in cerebellar stellate cells induced by pharmacological agents: Non-sequential spike adding.

Cerebellar stellate cells (CSCs) are spontaneously active, tonically firing (5-30 Hz), inhibitory interneurons that synapse onto Purkinje cells. We previously analyzed the excitability properties of CSCs, focusing on four key features: type I excitability, non-monotonic first-spike latency, switching in responsiveness and runup (i.e., temporal increase in excitability during whole-cell configuration). In this study, we extend this analysis by using whole-cell configuration to show that these neurons can also burst when treated with certain pharmacological agents separately or jointly. Indeed, treatment with 4-Aminopyridine (4-AP), a partial blocker of delayed rectifier and A-type K+ channels, at low doses induces a bursting profile in CSCs significantly different than that produced at high doses or when it is applied at low doses but with cadmium (Cd2+), a blocker of high voltage-activated (HVA) Ca2+ channels. By expanding a previously revised Hodgkin-Huxley type model, through the inclusion of Ca2+-activated K+ (K(Ca)) and HVA currents, we explain how these bursts are generated and what their underlying dynamics are. Specifically, we demonstrate that the expanded model preserves the four excitability features of CSCs, as well as captures their bursting patterns induced by 4-AP and Cd2+. Model investigation reveals that 4-AP is potentiating HVA, inducing square-wave bursting at low doses and pseudo-plateau bursting at high doses, whereas Cd2+ is potentiating K(Ca), inducing pseudo-plateau bursting when applied in combination with low doses of 4-AP. Using bifurcation analysis, we show that spike adding in square-wave bursts is non-sequential when gradually changing HVA and K(Ca) maximum conductances, delayed Hopf is responsible for generating the plateau segment within the active phase of pseudo-plateau bursts, and bursting can become "chaotic" when HVA and K(Ca) maximum conductances are made low and high, respectively. These results highlight the secondary effects of the drugs applied and suggest that CSCs have all the ingredients needed for bursting.

Checkpoint kinase­1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair.

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA­mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK­8, and comet assay. The results demonstrated that shRNA­induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.

Differential expression of the c-fos protein and synaptophysin in zebrin II positive and zebrin II negative cerebellar cortical areas in 4-aminopyridine seizures.

The present study examined temporal activation patterns of rat cerebellar cortical neurons in 4-aminopyridine induced seizures, using c-fos protein as a marker of neuronal activity. C-fos-containing cells were counted in each cerebellar cortical layer, and cell count was compared between zebrin II positive and zebrin II negative bands of the lobules of the vermis and cerebellar hemispheres. We found significant activation of granule cells and interneurons of the molecular layer in zebrin II positive bands. The Purkinje cells, in contrast, exhibited non-significant, scattered c-fos immunoreactivity across all bands. Fluctuation of synaptophysin expression in the mossy fibre rosettes of the granular layer was determined via light microscopic immunohistochemistry. We detected a transient, significant decrease in synaptophysin staining density following 4-aminopyridine seizures, which may indicate short-term synaptic depression. We also identified different timing of increased c-fos expression in the neurons of the cerebellar cortex in different cortical zones. In particular, the activation pattern of the interneurons of the molecular layer reflected the climbing fibre distribution, reflecting the zonal olivo-cortico-nuclear organization. Seizure-induced activation of the granule cells corresponded with the zebrin II positive zones. This observation raises the possibility that zebrin II positive compartments may be more susceptible to cerebellar convulsions.

The Neuropeptide Galanin Is Required for Homeostatic Rebound Sleep following Increased Neuronal Activity.

Sleep pressure increases during wake and dissipates during sleep, but the molecules and neurons that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole-brain mapping with the neuronal activity marker phosphorylated extracellular signal-regulated kinase (pERK) identified preoptic Galanin (Galn)-expressing neurons as selectively active during rebound sleep, and the relative induction of galn transcripts was predictive of total rebound sleep time. Galn is required for sleep homeostasis, as galn mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galn plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat.

4-Aminopyridine attenuates muscle atrophy after sciatic nerve crush injury in mice.

INTRODUCTION: We recently demonstrated the beneficial effects of 4-aminopyridine (4-AP), a potassium channel blocker, in enhancing remyelination and recovery of nerve conduction velocity and motor function after sciatic nerve crush injury in mice. Although muscle atrophy occurs very rapidly after nerve injury, the effect of 4-AP on muscle atrophy and intrinsic muscle contractile function is largely unknown. METHODS: Mice were assigned to sciatic nerve crush injury and no-injury groups and were followed for 3, 7, and 14 days with/without 4-AP or saline treatment. Morphological, functional, and transcriptional properties of skeletal muscle were assessed. RESULTS: In addition to improving in vivo function, 4-AP significantly reduced muscle atrophy with increased muscle fiber diameter and contractile force. Reduced muscle atrophy was associated with attenuated expression of atrophy-related genes and increased expression of proliferating stem cells. DISCUSSION: These findings provide new insights into the potential therapeutic benefits of 4-AP against nerve injury-induced muscle atrophy and dysfunction. Muscle Nerve 60: 192-201, 2019.

AMP-activated Protein Kinase Controls Immediate Early Genes Expression Following Synaptic Activation Through the PKA/CREB Pathway.

Long-term memory formation depends on the expression of immediate early genes (IEGs). Their expression, which is induced by synaptic activation, is mainly regulated by the 3',5'-cyclic AMP (cAMP)-dependent protein kinase/cAMP response element binding protein (cAMP-dependent protein kinase (PKA)/ cAMP response element binding (CREB)) signaling pathway. Synaptic activation being highly energy demanding, neurons must maintain their energetic homeostasis in order to successfully induce long-term memory formation. In this context, we previously demonstrated that the expression of IEGs required the activation of AMP-activated protein kinase (AMPK) to sustain the energetic requirements linked to synaptic transmission. Here, we sought to determine the molecular mechanisms by which AMPK regulates the expression of IEGs. To this end, we assessed the involvement of AMPK in the regulation of pathways involved in the expression of IEGs upon synaptic activation in differentiated primary neurons. Our data demonstrated that AMPK regulated IEGs transcription via the PKA/CREB pathway, which relied on the activity of the soluble adenylyl cyclase. Our data highlight the interplay between AMPK and PKA/CREB signaling pathways that allows synaptic activation to be transduced into the expression of IEGs, thus exemplifying how learning and memory mechanisms are under metabolic control.

Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on [Ca2+]i and behaviour of human sperm.

STUDY QUESTION: How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects on human sperm? SUMMARY ANSWER: Behaviour of human sperm (proportion of hyperactivated cells and motility kinematics) were related directly to [Ca2+]i irrespective of pH or the agonist applied. WHAT IS KNOWN ALREADY?: CatSper channels of human sperm, which are central to generation of sperm [Ca2+]i signals and induction of hyperactivated motility, are activated by intracellular alkalinization and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) (which mobilizes stored Ca2+ but also raises pHi) as an inducer of hyperactivation. STUDY DESIGN, SIZE, DURATION: This was a laboratory study, spanning ~18 months that used 15 sperm donors and involved more than 100 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (ERN_12-0570R). [Ca2+]i responses of suspended cell populations were examined by fluorimetric recording and motility parameters assessed by computer-assisted sperm analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing pHo from 7.4 to 8.5 raised pHi (from 6.9 to 7.2) and significantly increased both [Ca2+]i and the proportion of hyperactivated cells. Stimulation of cells with P4 (1 nM-20 µM) induced a biphasic (transient and plateau) increase in [Ca2+]i. The [Ca2+]i increase was of similar amplitude and dose-dependency at pHo = 7.4 and pHo = 8.5. 4-aminopyridine (0.2-5 mM) induced a biphasic [Ca2+]i increase that was dose-dependent across the entire range tested and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau phase of the progesterone and 4-AP-induced [Ca2+]i responses. Progesterone had only a small effect on hyperactivated motility even at the highest dose used (20 µM; < 5% increase in the proportion of cells classified as hyperactivated) which was insensitive to pHo. 4-Aminopyridine potently stimulated hyperactivated motility, this effect being dose-dependent and greatly enhanced at pHo = 8.5. The relationship between [Ca2+]i (fluorescence of fluo4) and proportion of hyperactivated cells, irrespective of pHo, agonist or dose, was fitted by a single curve (second order polynomial; R2 = 0.96). Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) showed a linear relationship to [Ca2+]i (R2 > 0.9). LIMITATIONS, REASONS FOR CAUTION: This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca2+]i transient may not have been reported by the dye. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that (i) activation of human sperm CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition of hyperactivated motility is not significantly enhanced by intracellular alkalinization; (ii) VCL, ALH and hyperactivation are directly related to [Ca2+]i, irrespective of the mechanism by which Ca2+ is mobilized, and the ability of stimuli to induce prolonged [Ca2+]i elevation (as occurs upon mobilization of stored Ca2+) determines the observed effect on cell behaviour. STUDY FUNDING/COMPETING INTEREST(S): CA was supported by the Nigerian government (Tertiary Education Trust (TET) Fund). The authors have no conflicts of interest.

Pharmacological characterization of crotamine effects on mice hind limb paralysis employing both ex vivo and in vivo assays: Insights into the involvement of voltage-gated ion channels in the crotamine action on skeletal muscles.

The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10-25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.

Regulation of P2X7 receptor function of neural progenitor cells in the hippocampal subgranular zone by neuronal activity in the dentate gyrus.

P2X7 receptors (Rs) mediate apoptosis/necrosis in neuronal and non-neuronal systems. Patch-clamp recordings from dentate gyrus (DG) granule cells in acutely prepared hippocampal slices of mice showed that incubation with 4-aminopyridine (4-AP) causes an excitability increase. This led to an enhanced sensitivity of P2X7Rs of the underlying subgranular zone neural progenitor cells (NPCs) towards dibenzoyl-ATP (Bz-ATP). The glutamatergic agonists NMDA and AMPA, as well as the purinergic agonist ATP also increased the Bz-ATP-induced current amplitudes (IBzATP). Tetrodotoxin as well as the standard antiepileptic drugs phenytoin, valproic acid and gabapentin counteracted the effect of 4-AP, most likely by decreasing the firing rate and/or action potential duration of DG granule cells and in consequence the release of ATP/glutamate onto NPCs. Experiments with organotypic hippocampal slice cultures confirmed these results also under conditions when 4-AP was applied for longer time periods and at much lower concentrations than used in acute slices. It was concluded that pathological firing modelled by 4-AP might trigger a sensitivity increase of P2X7Rs leading to necrosis/apoptosis of NPCs with the subsequent decrease of NPC, and in consequence, granule cell number. Hence, supersensitive P2X7Rs may exert a beneficial counter-regulatory effect by reducing the chances for the evolution of chronic temporal lobe epilepsy by ectopically located granule cells.

Modulation of hippocampal excitability via the hydroxycarboxylic acid receptor 1.

In addition to its prominent role as an energetic substrate in the brain, lactate is emerging as a signaling molecule capable of controlling neuronal excitability. The finding that the lactate-activated receptor (hydroxycarboxylic acid receptor 1; HCA1) is widely expressed in the brain opened up the possibility that lactate exerts modulation of neuronal activity via a transmembranal receptor-linked mechanism. Here, we show that lactate causes biphasic modulation of the intrinsic excitability of CA1 pyramidal cells. In the low millimolar range, lactate or the HCA1 agonist 3,5-DHBA reduced the input resistance and membrane time constant. In addition, activation of HCA1 significantly blocked the fast inactivating sodium current and increased the delay from inactivation to a conducting state of the sodium channel. As the observed actions occurred in the presence of 4-CIN, a blocker of the neuronal monocarboxylate transporter, the possibility that lactate acted via neuronal metabolism is unlikely. Consistently, modulation of the intrinsic excitability was abolished when CA1 pyramidal cells were dialyzed with pertussis toxin, indicating the dependency of a Gαi/o -protein-coupled receptor. The activation of HCA1 appears to serve as a restraining mechanism during enhanced network activity and may function as a negative feedback for the astrocytic production of lactate.