RESUMEN

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Asunto(s)

Venenos de Abeja , Abejas/genética , Animales , Perfilación de la Expresión Génica , Transcriptoma , Genómica , Duplicación de Gen

RESUMEN

Cognitive dysfunction induced by anesthesia in the infant is a crucial clinical issue that is still being debated and the focus of concern for the parents. However, the mechanism of cognitive decline caused by anesthesia and the corresponding treatment methods remain unclear. Postnatal day 7 (PND7) C57BL/6 mice included in the study were randomly divided into a control group (Control), a group with repeated exposure to sevoflurane (Sevo), and an Apamin intervention group (Sevo + Apamin). Apamin (0.5 µL at the concentration of 100 nmol/L) was injected into the bilateral hippocampus of mice. qRT-PCR, enzyme-linked immunosorbent assay (ELISA), and western blotting assay were used to evaluate the protein levels in the hippocampus. Object location memory (OLM) and novel object recognition (NOR) tasks, as well as elevated plus maze and contextual and cued fear conditioning tasks were used to evaluate the cognitive function of mice. Apamin mitigated sevoflurane-induced cognitive impairment of mice, sevoflurane-induced neuronal injury, and sevoflurane-induced activation of microglial in the hippocampus of the mice. Apamin inhibited M1-type polarization but promoted M2-type polarization of microglia after neonatal sevoflurane exposures in the hippocampus. In conclusion, Apamin attenuates neonatal sevoflurane exposures that cause cognitive deficits in mice through regulating hippocampal neuroinflammation.

Asunto(s)

Disfunción Cognitiva , Enfermedades Neuroinflamatorias , Animales , Ratones , Ratones Endogámicos C57BL , Apamina , Sevoflurano , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/tratamiento farmacológico , Cognición , Hipocampo

RESUMEN

Acetaminophen (APAP) overdose can cause severe liver damage, but therapeutic options are limited. Apamin is a natural peptide present in bee venom and has antioxidant and anti-inflammatory properties. Accumulating evidence suggests that apamin has favorable actions in rodent models of inflammatory disorders. Here, we examined the effect of apamin on APAP-evoked hepatotoxicity. Intraperitoneal administration of apamin (0.1 mg/kg) alleviated histological abnormalities and reduced serum levels of liver enzymes in mice injected with APAP. Apamin inhibited oxidative stress through an increase in the amount of glutathione and activation of the antioxidant system. Apamin also attenuated apoptosis with inhibition of caspase-3 activation. Moreover, apamin reduced serum and hepatic levels of cytokines in APAP-injected mice. These effects were accompanied by suppression of NF-κB activation. Furthermore, apamin inhibited chemokine expression and inflammatory cell infiltration. Our results suggest that apamin dampens APAP-evoked hepatotoxicity through inhibiting oxidative stress, apoptosis, and inflammation.

RESUMEN

BACKGROUND: Small-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. METHODS: Apamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF). RESULTS: ISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. CONCLUSIONS: ISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

Asunto(s)

Fibrilación Atrial , Animales , Humanos , Fibrilación Atrial/metabolismo , Apamina/metabolismo , Apamina/farmacología , Primaquina/metabolismo , Primaquina/farmacología , Calmodulina/metabolismo , Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Antiarrítmicos/uso terapéutico , Potenciales de Acción/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo

RESUMEN

Parkinson's disease (PD), a neurodegenerative disorder, is characterized by the loss of dopaminergic (DA) neurons. The pathogenesis of PD is associated with several factors including oxidative stress, inflammation, and mitochondrial dysfunction. Ca2+ signaling plays a vital role in neuronal signaling and altered Ca2+ homeostasis has been implicated in many neuronal diseases including PD. Recently, we reported that apamin (APM), a selective antagonist of the small-conductivity Ca2+-activated K+ (SK) channel, suppresses neuroinflammatory response. However, the mechanism(s) underlying the vulnerability of DA neurons were not fully understood. In this study, we investigated whether APM affected 1-methyl-4-phenyl pyridinium (MPP+)-mediated neurotoxicity in SH-SY5Y cells and rat embryo primary mesencephalic neurons. We found that APM decreased Ca2+ overload arising from MPP+-induced neurotoxicity response through downregulating the level of CaMKII, phosphorylation of ERK, and translocation of nuclear factor NFκB/signal transducer and activator of transcription (STAT)3. Furthermore, we showed that the correlation of MPP+-mediated Ca2+ overload and ERK/NFκB/STAT3 in the neurotoxicity responses, and dopaminergic neuronal cells loss, was verified through inhibitors. Our findings showed that APM might prevent loss of DA neurons via inhibition of Ca2+-overload-mediated signaling pathway and provide insights regarding the potential use of APM in treating neurodegenerative diseases.

Asunto(s)

Neuroblastoma , Fármacos Neuroprotectores , Síndromes de Neurotoxicidad , Enfermedad de Parkinson , Humanos , Ratas , Animales , Calcio/metabolismo , Apamina/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Fármacos Neuroprotectores/farmacología , Neuroblastoma/metabolismo , 1-Metil-4-fenilpiridinio/toxicidad , Neuronas Dopaminérgicas/metabolismo , Transducción de Señal , Estrés Oxidativo , Enfermedad de Parkinson/metabolismo , FN-kappa B/metabolismo , Síndromes de Neurotoxicidad/patología , Apoptosis , Línea Celular Tumoral

RESUMEN

Apamin is often cited as one of the few substances selectively acting on small-conductance Ca2+-activated potassium channels (KCa2). However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels (KCa, KV, NaV, nAChR, ASIC, and others) and confirmed its unique selectivity to KCa2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity KCa2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a KCa2-selective pharmacological tool and as a template for drug design.

RESUMEN

The discovery of new drugs has benefited significantly from the development of research in venomics, increasing our understanding of the envenomation processes. It has been previously reported that honeybee venom (HBV) exhibits several pharmacological activities such as anti-inflammatory, antibacterial, antimutagenic, radioprotective, and anticancer activity and may inclusively act as a complementary treatment for SARS-CoV-2. It composition consists mainly on melittin, phospholipase A2, and apamin but other constituents such as hyaluronidase, mast cell degranulating peptide and secapin are also relevant for its bioactivity. However, and because HBV is not officially recognized as a drug, until now, the international community did not establish quality standards for it. To uncover its exact composition, and boost the discovery of HBV-derived drugs, a significant number of techniques were developed. In this review, a relevant overview of the so far published analytical methods for HBV characterization is organized with the aim to accelerate its future standardization. The literature search was performed within PubMed, Google Scholar, and Science Direct by selecting specific documents and exploring HBV evaluation.

RESUMEN

Ellagic acid has recently attracted increasing attention regarding its role in the prevention and treatment of cancer. Surface functionalized nanocarriers have been recently studied for enhancing cancer cells' penetration and achieving better tumor-targeted delivery of active ingredients. Therefore, the present work aimed at investigating the potential of APA-functionalized emulsomes (EGA-EML-APA) for enhancing cytototoxic activity of EGA against human breast cancer cells. Phospholipon® 90 G: cholesterol molar ratio (PC: CH; X1, mole/mole), Phospholipon® 90 G: Tristearin weight ratio (PC: TS; X2, w/w) and apamin molar concentration (APA conc.; X3, mM) were considered as independent variables, while vesicle size (VS, Y1, nm) and zeta potential (ZP, Y2, mV) were studied as responses. The optimized formulation with minimized vs. and maximized absolute ZP was predicted successfully utilizing a numerical technique. EGA-EML-APA exhibited a significant cytotoxic effect with an IC50 value of 5.472 ± 0.21 µg/mL compared to the obtained value from the free drug 9.09 ± 0.34 µg/mL. Cell cycle profile showed that the optimized formulation arrested MCF-7 cells at G2/M and S phases. In addition, it showed a significant apoptotic activity against MCF-7 cells by upregulating the expression of p53, bax and casp3 and downregulating bcl2. Furthermore, NF-κB activity was abolished while the expression of TNfα was increased confirming the significant apoptotic effect of EGA-EML-APA. In conclusion, apamin-functionalized emulsomes have been successfully proposed as a potential anti-breast cancer formulation.

Asunto(s)

Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Apamina , Ácido Elágico/farmacología , Excipientes , Humanos , Lípidos , Células MCF-7 , Tamaño de la Partícula

RESUMEN

Diabetic hyperglycemia delays wound healing, leading to serious consequences. Topical antibiotics can reduce the risk of a wound infection during healing; nevertheless, the microbial fight against antibiotics brings about public health challenges. Anti-microbial peptides (AMPs) belong to a novel class of drug that is used to prevent and treat systemic and topical infections. The aim of the current work was to achieve better wound healing in diabetic rats by conjugating the anti-microbial peptide "apamin" (APA) with the broad-spectrum antibiotic "ceftriaxone" (CTX) to form a nanocomplex. The CTX-APA nanoconjugate formulation was optimized using a Box-Behnken design. The optimized CTX-APA nanoconjugate formulation was evaluated for its size and zeta potential, and was then examined using transmission electron microscopy (TEM). The CTX-APA nanoconjugate was loaded onto a hydroxypropyl methylcellulose (2% w/v)-based hydrogel. It was observed that the application of the CTX-APA nanocomplex on the wounded skin of diabetic rats accelerated the regeneration of the epithelium, granulation tissue formation, epidermal proliferation, and keratinization. The nanocomplex was capable of significantly reducing the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), while increasing the expression of transforming growth factor beta-1 (TGF-ß1) as well as the angiogenic markers: hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Conclusively, the application of an ion-paired CTX-APA nanocomplex enhances wound healing in diabetic rats.

RESUMEN

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 µM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 µM DCEBIO), but not by an IKCa channel opener (1 µM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 µM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Asunto(s)

Melatonina , Glándula Pineal , Canales de Potasio Calcio-Activados , Animales , Apamina/farmacología , Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Melatonina/metabolismo , Glándula Pineal/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Pirazoles/farmacología , Ratas , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo

RESUMEN

Modern venomics is increasing its focus on hymenopterans such as honeybees, bumblebees, parasitoid wasps, ants and true wasps. However solitary bees remain understudied in comparison and the few available venom studies focus on short melittin-like sequences and antimicrobial peptides. Herein we describe the first comprehensive venom profile of a solitary bee, the violet carpenter bee Xylocopa violacea, by using proteo-transcriptomics. We reveal a diverse and complex venom profile with 43 different protein families identified from dissected venom gland extracts of which 32 are also detected in the defensively injected venom. Melittin and apamin are the most highly secreted components, followed by Phospholipase A2, Icarapin, Secapin and three novel components. Other components, including eight novel protein families, are rather lowly expressed. We further identify multiple forms of apamin-like peptides. The melittin-like sequences of solitary bees separate into two clades, one comprised most sequences from solitary bees including xylopin (the variant in Xylocopa), while sequences from Lasioglossa appear closer related to melittin-like peptides from Bombus (Bombolittins). Our study suggests that more proteo-transcriptomic data from other solitary bees should be complemented with corresponding genome data to fully understand the evolution and complexity of bee venom proteins, and is of a particular need to disentangle the ambiguous phylogenetic relations of short peptides.

RESUMEN

We have previously reported in a feline model of acute peripheral vestibulopathy (APV) that the sudden, unilateral, and irreversible loss of vestibular inputs induces selective overexpression of small conductance calcium-activated potassium (SK) channels in the brain stem vestibular nuclei. Pharmacological blockade of these ion channels by the selective antagonist apamin significantly alleviated the evoked vestibular syndrome and accelerated vestibular compensation. In this follow-up study, we aimed at testing, using a behavioral approach, whether the antivertigo (AV) effect resulting from the antagonization of SK channels was species-dependent or whether it could be reproduced in a rodent APV model, whether other SK channel antagonists reproduced similar functional effects on the vestibular syndrome expression, and whether administration of SK agonist could also alter the vestibular syndrome. We also compared the AV effects of apamin and acetyl-DL-leucine, a reference AV compound used in human clinic. We demonstrate that the AV effect of apamin is also found in a rodent model of APV. Other SK antagonists also produce a trend of AV effect when administrated during the acute phase of the vertigo syndrome. Conversely, the vertigo syndrome is worsened upon administration of SK channel agonist. It is noteworthy that the AV effect of apamin is superior to that of acetyl-DL-leucine. Taken together, these data reinforce SK channels as a pharmacological target for modulating the manifestation of the vertigo syndrome during APV.

RESUMEN

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system presented by autoimmune manifestations. This study aimed at investigating the effects of apamin administration on the activated T cell population in an experimental autoimmune encephalomyelitis (EAE) MS model. Thirty mice underwent EAE induction and were then randomly divided into 5 groups. Three groups received 10, 50, and 100 µg/kg apamin; the fourth group received 1 mg/kg dexamethasone; and the fifth group received the equivalent amount of PBS (phosphate-buffered saline) intraperitoneally. Peripheral CD4 + cell and memory T cell distribution was measured with a flow cytometer every week. Also, CD4 + and CD8 + cell infiltration to the brain was assessed with immunohistochemistry. It was observed that the group receiving 50 µg/kg apamin had a lower EAE score in comparison with the groups receiving 100 µg/kg apamin (p 0.014). Also, peripheral blood memory cells with CD44 + , CD62L - , and CD4 + markers were decreased in apamin-administered groups. Regarding the infiltrated CD8 + cells, a significant decrease (p 0.002) was observed in the group receiving 50 µg/kg apamin compared with the control group. These results indicate that 50-µg/kg doses of apamin had an effective treatment over 14 days; it reduced both the severity of symptoms and the infiltration of CD8 + cells into the CNS. Moreover, it increased myelin density and decreased the circulation of CD62L - , CD44L - , and CD44 + memory T cells. So, it appears that apamin plays a critical role in regulating immunity and reducing the complications of autoimmune MS.

Asunto(s)

Apamina/uso terapéutico , Barrera Hematoencefálica , Esclerosis Múltiple/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Animales , Apamina/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Dexametasona/uso terapéutico , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/etiología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Vaina de Mielina/patología

RESUMEN

Apamin is a minor component of bee venom and is a polypeptide with 18 amino acid residues. Although apamin is considered a neurotoxic compound that blocks the potassium channel, its neuroprotective effects on neurons have been recently reported. However, there is little information about the underlying mechanism and very little is known regarding the toxicological characterization of other compounds in bee venom. Here, cultured mature cortical neurons were treated with bee venom components, including apamin, phospholipase A2, and the main component, melittin. Melittin and phospholipase A2 from bee venom caused a neurotoxic effect in dose-dependent manner, but apamin did not induce neurotoxicity in mature cortical neurons in doses of up to 10 µg/mL. Next, 1 and 10 µg/mL of apamin were applied to cultivate mature cortical neurons. Apamin accelerated neurite outgrowth and axon regeneration after laceration injury. Furthermore, apamin induced the upregulation of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated gene expression in mature cortical neurons. Due to its neurotherapeutic effects, apamin may be a promising candidate for the treatment of a wide range of neurological diseases.

Asunto(s)

Apamina/farmacología , Venenos de Abeja/farmacología , Enfermedades Cerebelosas/tratamiento farmacológico , Laceraciones/tratamiento farmacológico , Regeneración Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Modelos Animales de Enfermedad , Humanos , Ratas , Ratas Sprague-Dawley

RESUMEN

Pancreatic cancer has a low survival rate and has limited therapeutic options due to the peculiarity of the tumor tissue. Cancer nanotechnology provides several opportunities to resolve such difficulties as a result of the high surface-to-volume ratio of nanostructures. Peptide-drug nanocomplexes have proved to have immense potential in anticancer activity against pancreatic cancer cells. Thus, in the present study apamin (APA) and alendronate sodium (ALS) were combined to form nanocomplexes (APA-ALS-NC) against pancreatic cancer cells. Optimization of ALS, incubation time, and sonication time in terms of particle size of the nanocomplex was carried out. The optimized formulation was evaluated for anticancer activities in pancreatic cancer cells (PANC-1 cells). A Box-Behnken design using ALS, incubation time, and sonication time as independent factors and particle size as the response was chosen to optimize the APA-ALS-NC formulation. The optimized APA-ALS-NC had a particle size of 161.52 ± 8.4 nm. The evaluation of APA-ALS-NC in PANC-1 cells was carried out using various in vitro tests. The IC50 values were determined by MTT assay and found to be 37.6 ± 1.65, 13.4 ± 0.59, and 1.01 ± 0.04 µg/mL for ALS, APA, and APA-ALS-NC, respectively. The higher cytotoxicity activity of APA-ALS-NC was confirmed from the higher percentage of cells in the necrosis phase (apoptosis study) and the G2-M phase (cell cycle study) compared to that of ALS and APA. While the loss of mitochondrial membrane potential was less for APA-ALS-NC, the levels of IL-1ß, TNF-α, caspase-3, ROS, IL-6, and NF-kB showed that APA-ALS-NC can significantly enhance apoptosis and cytotoxicity in PANC-1 cells. Moreover, Bax (10.87 ± 1.36), Bcl-2 (0.27 ± 0.02), and p53 (9.16 ± 1.22) gene expressions confirmed that APA-ALS-NC had a significant apoptotic effect compared to ALS and APA. In summary, the APA-ALS-NC had a more significant cytotoxic effect than ALS and APA. The results of the present study are promising for further evaluation in pre-clinical and clinical trials for arriving at a successful therapeutic strategy against pancreatic cancer.

RESUMEN

BACKGROUND AND PURPOSE: KATP channels are negative regulators of lymphatic vessel excitability and contractility and are proposed to be targets for immune cell products that inhibit lymph transport. Previous studies in rat and guinea pig mesenteric lymphatics found that NO-mediated inhibition of lymphatic contraction was prevented or reversed by the KATP channel inhibitor, glibenclamide. We revisited this hypothesis using mouse lymphatic vessels and KATP channel knockout mice. EXPERIMENTAL APPROACH: Mouse popliteal lymphatics were isolated, and contractility was assessed using pressure myography. K+ channel expression was determined by PCR analysis of FACS-purified lymphatic smooth muscle cells. KEY RESULTS: The NO-producing agonist, ACh, and the NO donor, NONOate, both produced dose-dependent inhibition of spontaneous lymphatic contractions that were blocked by the soluble GC inhibitor, ODQ, or the PKG inhibitor, Rp-8-Br-PET-cGMPS. Surprisingly, the inhibitory effects of both were preserved in Kir 6.1-/- vessels, suggesting that KATP channels did not mediate NO-induced responses. We hypothesized a role for BK channels, given their prominence in arterial smooth muscle. Indeed, BK channels were expressed in mouse lymphatic smooth muscle and NS11021 (a BK channel activator) caused dilation and reduced contraction frequency, whereas iberiotoxin and penitrem A (BK channel inhibitors) produced right-ward shifts in NONOate concentration-response curves. CONCLUSION AND IMPLICATIONS: Inhibition of mouse lymphatic contractions by NO primarily involves activation of BK channels, rather than KATP channels. Thus, BK channels are a potential target for therapeutic reversal of lymph pump inhibition by NO generated by immune cell activation of iNOS in chronic lymphoedema.

Asunto(s)

Calcio , Óxido Nítrico , Adenosina Trifosfato , Animales , Calcio/metabolismo , Cobayas , Canales KATP , Canales de Potasio de Gran Conductancia Activados por el Calcio , Ratones , Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Ratas

RESUMEN

Background: Small-conductance Ca2+-activated K+ channels (SK channels) have been proposed as antiarrhythmic targets for the treatment of atrial fibrillation. We previously demonstrated that the 5-HT3 receptor antagonist ondansetron inhibits heterologously expressed, human SK2 (hSK2) currents as well as native cardiac SK currents in a physiological extra-/intracellular [K+] gradient at therapeutic (i.e., sub-micromolar) concentrations. A recent study, using symmetrical [K+] conditions, challenged this result. The goal of the present study was to revisit the inhibitory effect of ondansetron on hSK2-mediated currents in symmetrical [K+] conditions. Experimental Approach: The whole-cell patch clamp technique was used to investigate the effects of ondansetron and apamin on hSK2-mediated currents expressed in HEK 293 cells. Currents were measured in symmetrical [K+] conditions in the presence of 100 nM [Ca2+]o. Results: Expression of hSK2 produced inwardly rectifying whole-cell currents in the presence of 400 nM free cytosolic Ca2+. Ondansetron inhibited whole-cell hSK2 currents with IC 50 values of 154 and 113 nM at -80 and 40 mV, respectively. Macroscopic current inhibited by ondansetron and current inhibited by apamin exhibited inwardly rectifying current-voltage relationships with similar reversal potentials (apamin, ∼5 mV and ondansetron, ∼2 mV). Ondansetron (1 µM) in the continuing presence of apamin (100 nM) had no effect on hSK2-mediated whole-cell currents. Wild-type HEK 293 cells did not express ondansetron- or apamin-sensitive currents. Conclusion: Ondansetron in sub-micromolar concentrations inhibits hSK2 currents even under altered ionic conditions.

RESUMEN

Renal fibrosis is a progressive and chronic process that influences kidneys with chronic kidney disease (CKD), irrespective of cause, leading to irreversible failure of renal function and end-stage kidney disease. Among the signaling related to renal fibrosis, transforming growth factor-ß1 (TGF-ß1) signaling is a major pathway that induces the activation of myofibroblasts and the production of extracellular matrix (ECM) molecules. Apamin, a component of bee venom (BV), has been studied in relation to various diseases. However, the effect of apamin on renal interstitial fibrosis has not been investigated. The aim of this study was to estimate the beneficial effect of apamin in unilateral ureteral obstruction (UUO)-induced renal fibrosis and TGF-ß1-induced renal fibroblast activation. This study revealed that obstructive kidney injury induced an inflammatory response, tubular atrophy, and ECM accumulation. However, apamin treatment suppressed the increased expression of fibrotic-related genes, including α-SMA, vimentin, and fibronectin. Administration of apamin also attenuated the renal tubular cells injury and tubular atrophy. In addition, apamin attenuated fibroblast activation, ECM synthesis, and inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 by suppressing the TGF-ß1-canonical and non-canonical signaling pathways. This study showed that apamin inhibits UUO-induced renal fibrosis in vivo and TGF-ß1-induced renal fibroblasts activation in vitro. Apamin inhibited the inflammatory response, tubular atrophy, ECM accumulation, fibroblast activation, and renal interstitial fibrosis through suppression of TGF-ß1/Smad2/3 and STAT3 signaling pathways. These results suggest that apamin might be a potential therapeutic agent for renal fibrosis. KEY MESSAGES: UUO injury can induce renal dysfunction; however, apamin administration prevents renal failure in UUO mice. Apamin inhibited renal inflammatory response and ECM deposition in UUO-injured mice. Apamin suppressed the activation of myofibroblasts in vivo and in vitro. Apamin has the anti-fibrotic effect on renal fibrosis via regulation of TGF-ß1 canonical and non-canonical signaling.

Asunto(s)

Antiinflamatorios/farmacología , Antifibróticos/farmacología , Apamina/farmacología , Enfermedades Renales/prevención & control , Riñón/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Obstrucción Ureteral/tratamiento farmacológico , Animales , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Ratones Endogámicos C57BL , Fosforilación , Ratas , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología

RESUMEN

Multiple sclerosis is a chronic demyelinating disease with a functional disturbance in the immune system and axonal damages. It was shown that Apamin as a blood-brain barrier shuttle acts as a Ca2+ activated K+ channels (SK channels) blocker. In this study, the effects of Apamin on oligodendrocyte differentiation markers were evaluated on an induced model of MS. Briefly, C57BL/6 male mice (22 ± 5 g) except the control group were fed with 0.2% (w/w) cuprizone pellets for 6 weeks. After cuprizone withdrawal, mice were divided randomly into six groups. Apamin (100 µg/kg/BW) was administered intraperitoneally as a co-treatment during phase I (demyelination) or post-treatment phase II (remyelination) twice a week. Mice were anesthetized, perfused with phosphate-buffered saline, then fixed brains were coronally sectioned and the changes in oligodendrocytes markers such as Olig2, PDGFR-α, and BrdU incorporation were assessed by immunohistochemistry assay. Apamin administration increased Olig2+ cells in phase I as compared to the control group (p < 0.0001). Also, a decreasing trend in PDGFRa+ cells observed after cuprizone withdrawal (p < 0.001). 5-Bromo-2'-deoxyuridine (BrdU) incorporation test was confirmed stimulation of oligodendrocyte progenitor cell proliferation in phase I in the Apamin exposed group (p < 0.0001), especially at the subventricular zone. This study highlights the potential therapeutic effects of Apamin as a bee venom-derived peptide on oligodendrocyte precursor proliferation and elevation in myelin content in an oxidative induced multiple sclerosis model due to cuprizone exposure.

Asunto(s)

Venenos de Abeja/uso terapéutico , Barrera Hematoencefálica/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cuprizona/toxicidad , Esclerosis Múltiple/tratamiento farmacológico , Oligodendroglía/efectos de los fármacos , Animales , Venenos de Abeja/farmacología , Barrera Hematoencefálica/química , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proliferación Celular/fisiología , Quelantes/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos/análisis , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Oligodendroglía/química , Oligodendroglía/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/análisis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo

RESUMEN

Small-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

Asunto(s)

Cardiopatías/metabolismo , Corazón/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Animales , Humanos