Search Results (52)

B-raf (rapidly accelerated fibrosarcoma) is a crucial player within the ERK/MAPK signaling pathway. In the CNS, B-raf has been implicated in neuronal differentiation, long-term memory, and major depression. Mice with forebrain neuron-specific B-raf knockout show behavioral deficits in spatial learning tasks and impaired hippocampal long-term potentiation (LTP). To elucidate the mechanism(s) underlying diminished synaptic plasticity in B-raf-deficient mice, we performed whole-cell recordings from CA1 pyramidal cells in hippocampal slices of control and B-raf mutant mice. We found that the NMDA/AMPA ratio of excitatory postsynaptic currents (EPSCs) at the Schaffer collateral—CA1 pyramidal cell synapses was significantly reduced in B-raf mutants, which would at least partially account for their impaired LTP. Interestingly, the reduced NMDA component of field postsynaptic potentials in mutant preparations was partially reinstated by blocking the apamin-sensitive small-conductance Ca²⁺-activated K⁺ (SK) channels, which have also been reported to modulate hippocampal LTP and learning tasks. To determine the impact of B-raf-dependent signaling on SK current, we isolated the apamin-sensitive tail current after a strong depolarizing event and found indeed a significantly bigger SK current in B-raf-deficient cells compared to controls, which is consistent with the reduced action potential firing and the stronger facilitating effect of apamin on CA1 somatic excitability in B-raf-mutant hippocampus. Our data suggest that B-raf signaling readjusts the delicate balance between NMDA receptors and SK channels to promote synaptic plasticity and facilitate hippocampal learning and memory. Full article

S-nitrosoglutathione (GSNO), a promising S-nitrosothiol, has been recognized for its ability to modulate vascular tone through its vasodilatory, antiplatelet, and antiproliferative effects. However, data on its vasodilatory effects in human vessels remain limited, and its mechanisms of action have yet to be fully elucidated. In this study, we aimed to investigate the vasorelaxant effect of GSNO and its underlying mechanisms, with particular focus on the soluble guanylate cyclase (sGC)/nitric oxide (NO) pathway and potassium channels in isolated human saphenous veins (SVs) obtained from patients undergoing coronary artery bypass grafting (CABG). GSNO (10⁻⁸–10⁻⁴ M) produced concentration-dependent relaxations in SV rings precontracted with phenylephrine. These relaxations were unaffected by NO synthase inhibition with L-NAME (10⁻⁴ M, 30 min) or NO scavenging with PTIO (10⁻⁴ M, 30 min), but were significantly reduced by the sGC inhibitor, ODQ (10⁻⁵ M, 30 min). Inhibition of ATP-sensitive (glibenclamid; 10⁻⁵ M, 30 min.), high-conductance Ca²⁺-activated (charybdotoxin; 10⁻⁷ M, 30 min), small-conductance Ca²⁺-activated (apamin; 10⁻⁶ M, 30 min), or voltage-dependent (4-aminopyridine; 10⁻³ M, 30 min) potassium channels did not alter the maximum relaxant responses to GSNO. Furthermore, pretreatment with GSNO (10⁻⁴ M, 30 min) significantly attenuated both the contractile response and sensitivity to phenylephrine. Collectively, these findings demonstrate that GSNO exerts acute vasorelaxant and modulatory effects in human SV primarily via cGMP-dependent mechanisms, highlighting its potential as a local therapeutic agent for preventing graft spasm in CABG. Full article

Background/Objectives: Carvacrol is a naturally occurring phenolic monoterpene that is one of the main constituents of the essential oils of oregano (Origanum vulgare) and other herbs. Carvacrol has anti-inflammatory and antinociceptive effects. Carvacrol can activate and inhibit several second messengers and ionic channels at the systemic level. However, there is no evidence of the peripheral antinociception of carvacrol and its mechanism of action. This study was designed to determine whether the opioid receptor-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-K⁺ channel pathway is involved in the local antinociception of carvacrol. Methods: Wistar rats were injected with 1% formalin subcutaneously on the dorsal surface of the right hind paw with the vehicle or carvacrol (100–300 µg/paw). To determine whether the opioid receptor-NO-cGMP-K⁺ channel pathway and a biguanide-dependent mechanism are responsible for the local antinociception induced by carvacrol, the effect of the injection (10 min before the 1% formalin injection) with the corresponding vehicles, metformin, naltrexone, NG-L-nitro-arginine methyl ester (L-NAME), 1 H-(1,2,4)-oxadiazolo (4,2-a) quinoxalin-1-one (ODQ), and K⁺ channel blockers on the antinociception induced by local carvacrol (300 µg/paw) was determined. Results: In both phases of the formalin test, carvacrol produced antinociception. Naltrexone, metformin, L-NAME, ODQ, glibenclamide and glipizide (both ATP-sensitive K⁺ channel blockers), tetraethylammonium and 4-aminopyridine (voltage-gated K⁺ channel blockers), and apamin and charybdotoxin (Ca²⁺-activated K⁺ channel blockers) reversed the carvacrol-induced peripheral antinociception. Conclusions: The local peripheral administration of carvacrol produced significant antinociception and activated the opioid receptor-NO-cGMP-K⁺ channel pathway. Full article

Small conductance Ca²⁺-activated K⁺ (SK) channels are expressed in atria and ventricles. However, the data on the contribution of SK channels to atrial action potential (AP) repolarization are inconsistent. We investigated the effect of SK channel modulators on AP morphology in rabbit atrial myocytes and tested the hypothesis that pharmacological activation of SK channels suppresses pacing-induced Ca²⁺ transient (CaT) and AP duration (APD) alternans. At the cellular level, alternans are observed as beat-to-beat alternations in contraction, APD, and CaT amplitude, representing a risk factor for arrhythmias, including atrial fibrillation. Our results show that SK channel inhibition by apamin did not affect atrial APD under basal conditions. However, SK channel activation by NS309 significantly shortened APD, indicating the expression of functional SK channels. Moreover, the activation of SK channels reduced CaT amplitude and sarcoplasmic reticulum Ca²⁺ load. Activation of SK channels also suppressed pacing-induced CaT and APD alternans. K_V7.1 potassium channel inhibition, simulating long QT syndrome type-1 conditions, increased the risk of atrial CaT alternans, which was abolished by the activation of SK channels. In summary, our data suggest that pharmacological modulation of SK channels can potentially reduce atrial arrhythmia risk arising from pathological APD prolongation. Full article

Previous observations indicating a lower incidence of various types of cancer in beekeepers suggest that greater exposure to stings reduces the risk of cancer development. However, it is not known which of the active compounds of the bee venom (BV) may be responsible for the observed properties. The aim of this study is to evaluate the anti-glioblastoma effect of the main BV fractions. In addition, the effect of BV fractions on the activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) was assessed. Commercially available BV was divided into three fractions containing one of the main BV components: apamin (fraction #1), phospholipase A₂ (fraction #2), or melittin (fraction #3). The viability of glioblastoma lines (LN18 and LN229) compared to a physiological line (human MO3.13) was assessed using the MTT. MMP-2 and MMP-9 activity was assessed using gelatin zymography. Tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) levels in cell culture media were measured with the ELISA method. The fraction containing apamin did not show cytotoxic activity up to a concentration of 100 µg/mL. The fraction containing phospholipase A₂ partially reduced the cells’ viability at a concentration of 100 µg/mL. The greatest activity was demonstrated by the melittin-containing fraction which completely reduced the viability of glioma cells from a concentration of 2.5 μg/mL and inhibited the activity of the assessed metalloproteinases in a dose-dependent manner. After 72 h of incubation, the highest concentrations of TIMP-1 and TIMP-2 (approximately 150 ng/mL and 100 ng/mL, respectively) were observed in the LN229 line. In all tested lines, fraction #3, crude BV, and melittin reduced the secretion of both inhibitors into the medium in a dose-dependent manner. The melittin-containing fraction possessed anti-glioma properties in vitro, suggesting that melittin may be the main anticancer compound of BV. Full article

Aging is associated with decreased neuronal sensitivity and activity that creates deficits in cognitive processes, including learning, memory, motivation, general activity, and other behaviors. These effects are due in part to decreased intracellular Ca²⁺ homeostasis, increasing hyperpolarization of the resting potential in aged neurons and therefore decreasing their excitability. To reduce hyperpolarization in aged mice, we used apamin, a selective small conductance Ca²⁺-activated K⁺ (sK_Ca) channel blocker. By blocking the sK_Ca channels, apamin decreases the egress of the K⁺ out of the cell, reducing its hyperpolarization and causing it to be closer to threshold potential. As a result, neurons should be more sensitive to excitatory stimuli and more active. We evaluated the performance of aged mice in a selection of cognitive and behavioral tests prior to and after systemic applications of apamin or the vehicle saline. Apamin improved performance in short-term memory, increased attention to tasks, and decreased anhedonia. Apamin had no significant effect on long-term spatial and recognition memory, risk-taking behavior, sociability, and anxiety. Our results are compatible with the known effects of sK_Ca channel blockade on neuronal sensitivity and activity; however, these short-term effects were not reflected in longer-term alterations of neural plasticity responsible for long-term spatial and recognition memory or other more complex cognitive processes we evaluated. Full article

Background: Apigenin (4′,5,7-trihydroxyflavone), a flavonoid with potential cardiovascular benefits, has unclear mechanisms of action. This study investigates its effects on vascular function in Spontaneously Hypertensive Rats (SHRs). Methods: Mesenteric vascular beds (MVBs) were isolated from SHRs and perfused with increasing doses of apigenin after pre-contraction with phenylephrine. To explore the mechanisms, different MVBs were pre-perfused with antagonists and inhibitors, including indomethacin, L-NAME, and potassium channel blockers (tetraethylammonium, a non-specific potassium channel blocker; glibenclamide, an ATP-sensitive potassium channel blocker; 4-aminopyridine, a voltage-gated potassium channel blocker; charybdotoxin a selective intermediate-conductance calcium-activated potassium channel blocker; and apamin, a selective small-conductance calcium-activated potassium channel blocker). Results: Apigenin induced a dose-dependent reduction in perfusion pressure in MVBs with intact endothelium, an effect abolished by endothelium removal. L-NAME reduced apigenin-induced vasodilation by approximately 40%. The vasodilatory effect was blocked by potassium chloride and tetraethylammonium. The inhibition of small and intermediate calcium-activated potassium channels with charybdotoxin and apamin reduced apigenin-induced vasodilation by 50%, and a combination of these blockers with L-NAME completely inhibited the effect. Conclusions: Apigenin promotes vasodilation in resistance arteries through endothelial nitric oxide and calcium-activated potassium channels. These findings suggest that apigenin could have therapeutic potential in cardiovascular disease, warranting further clinical research. Full article

Maurotoxin (MTX) is a 34-residue peptide from Scorpio maurus venom. It is reticulated by four disulfide bridges with a unique arrangement compared to other scorpion toxins that target potassium (K⁺) channels. Structure–activity relationship studies have not been well performed for this toxin family. The screening of Scorpio maurus venom was performed by different steps of fractionation, followed by the ELISA test, using MTX antibodies, to isolate an MTX-like peptide. In vitro, in vivo and computational studies were performed to study the structure–activity relationship of the new isolated peptide. We isolated a new peptide designated MTX1, structurally related to MTX. It demonstrated toxicity on mice eight times more effectively than MTX. MTX1 blocks the Kv1.2 and Kv1.3 channels, expressed in Xenopus oocytes, with IC₅₀ values of 0.26 and 180 nM, respectively. Moreover, MTX1 competitively interacts with both ¹²⁵I-apamin (IC₅₀ = 1.7 nM) and ¹²⁵I-charybdotoxin (IC₅₀ = 5 nM) for binding to rat brain synaptosomes. Despite its high sequence similarity (85%) to MTX, MTX1 exhibits a higher binding affinity towards the Kv1.2 and SKCa channels. Computational analysis highlights the significance of specific residues in the β-sheet region, particularly the R27, in enhancing the binding affinity of MTX1 towards the Kv1.2 and SKCa channels. Full article

Apitoxin therapy (BVT—bee venom therapy) is an emerging complementary treatment utilizing bee venom for various medical conditions. This review explores the potential and therapeutic mechanisms of bee venom, focusing on its chemical composition and the methods for its extraction and purification to enhance safety while maintaining bioactivity. Bee venom contains amphipathic peptides such as melittin and apamin, enzymes like phospholipase A2, and bioamines including histamine and catecholamines, contributing to its pleiotropic effects. The therapeutic applications of bee venom span anti-inflammatory, analgesic, antimicrobial, antiviral, neuroprotective, anti-arthritic, and anti-cancer activities. Clinical and laboratory studies have demonstrated its efficacy in treating chronic and autoimmune diseases, pain management, and improving quality of life. The immunogenic properties of bee venom necessitate ongoing research to mitigate allergic reactions, ensuring its safe and effective use in medical practice. This review summarizes the current state of research on bee venom therapy, highlighting its potential benefits and future research directions. Full article

Neuroplasticity in the amygdala and its central nucleus (CeA) is linked to pain modulation and pain behaviors, but cellular mechanisms are not well understood. Here, we addressed the role of small-conductance Ca²⁺-activated potassium (SK) channels in pain-related amygdala plasticity. The facilitatory effects of the intra-CeA application of an SK channel blocker (apamin) on the pain behaviors of control rats were lost in a neuropathic pain model, whereas an SK channel activator (NS309) inhibited pain behaviors in neuropathic rats but not in sham controls, suggesting the loss of the inhibitory behavioral effects of amygdala SK channels. Brain slice electrophysiology found hyperexcitability of CeA neurons in the neuropathic pain condition due to the loss of SK channel-mediated medium afterhyperpolarization (mAHP), which was accompanied by decreased SK2 channel protein and mRNA expression, consistent with a pretranscriptional mechanisms. The underlying mechanisms involved the epigenetic silencing of the SK2 gene due to the increased DNA methylation of the CpG island of the SK2 promoter region and the change in methylated CpG sites in the CeA in neuropathic pain. This study identified the epigenetic dysregulation of SK channels in the amygdala (CeA) as a novel mechanism of neuropathic pain-related plasticity and behavior that could be targeted to control abnormally enhanced amygdala activity and chronic neuropathic pain. Full article

Beekeeping provides products with nutraceutical and pharmaceutical characteristics. These products are characterized by abundance of bioactive compounds. For different reasons, honey, royal jelly, propolis, venom, and pollen are beneficial to humans and animals and could be used as therapeutics. The pharmacological action of these products is related to many of their constituents. The main bioactive components of honey include oligosaccharides, methylglyoxal, royal jelly proteins (MRJPs), and phenolics compounds. Royal jelly contains jelleins, royalisin peptides, MRJPs, and derivatives of hydroxy-decenoic acid, particularly 10-hydroxy-2-decenoic acid (10-HDA), which possess antibacterial, anti-inflammatory, immunomodulatory, neuromodulatory, metabolic syndrome-preventing, and anti-aging properties. Propolis has a plethora of activities that are referable to compounds such as caffeic acid phenethyl ester. Peptides found in bee venom include phospholipase A2, apamin, and melittin. In addition to being vitamin-rich, bee pollen also includes unsaturated fatty acids, sterols, and phenolics compounds that express antiatherosclerotic, antidiabetic, and anti-inflammatory properties. Therefore, the constituents of hive products are particular and different. All of these constituents have been investigated for their properties in numerous research studies. This review aims to provide a thorough screening of the bioactive chemicals found in honeybee products and their beneficial biological effects. The manuscript may provide impetus to the branch of unconventional medicine that goes by the name of apitherapy. Full article

The roots of Gentiana lutea L. are utilized in the preparation of various beverages and herbal remedies, serving as a traditional remedy for gastrointestinal ailments. The spasmolytic activity that could substantiate the traditional use of G. lutea root had not been investigated. The main objective goal of the study was to determine the validity of its use as a traditional remedy. The extraction of G. lutea root was performed using a 50% hydroethanolic solvent with three different extraction techniques: ultrasound-assisted extraction (UAE), heat-assisted extraction, and percolation. The spasmolytic activity was tested on isolated rat ileum. The mechanism of action was monitored using the models of spontaneous contractions and acetylcholine-, histamine-, CaCl₂-, Bay K8644-, L-NAME-, ODQ-, apamin-, BaCl₂-, charybdotoxin-, glibenclamide-, TRAM-34-, and quinine-modified contractions. UAE, having the best bioactivity, was further subjected to a liquid–liquid extraction fractionation. HPLC phytochemical analysis was performed for all tested extracts and fractions. Gentian root extracts were rich in secoiridoids, xanthones, and flavonoids. The UAE has shown better results on spontaneous contractions in comparison to its fractions, leading to the more detailed testing of its spasmolytic mechanism of activity. The extract’s activity is primarily mediated through intermediate conductance Ca²⁺-activated K⁺ channels, ATP-sensitive K⁺ channels, voltage-sensitive K⁺ channels, and mechanisms that activate Ca²⁺ channels. Overall, the G. lutea root shows great potential in the treatment of spasmodic gastrointestinal ailments. Full article

Transient receptor potential (TRP) channels are pivotal in modulating vascular functions. In fact, topical application of cinnamaldehyde or capsaicin (TRPA1 and TRPV1 channel agonists, respectively) induces “local” changes in blood flow by releasing vasodilator neuropeptides. We investigated TRP channels’ contributions and the pharmacological mechanisms driving vasodilation in human isolated dermal arteries. Ex vivo studies assessed the vascular function of artery segments and analyzed the effects of different compounds. Concentration–response curves to cinnamaldehyde, pregnenolone sulfate (PregS, TRPM3 agonist), and capsaicin were constructed to evaluate the effect of the antagonists HC030031 (TRPA1); isosakuranetin (TRPM3); and capsazepine (TRPV1). Additionally, the antagonists/inhibitors olcegepant (CGRP receptor); L-NAME (nitric oxide synthase); indomethacin (cyclooxygenase); TRAM-34 plus apamin (K⁺ channels); and MK-801 (NMDA receptors, only for PregS) were used. Moreover, CGRP release was assessed in the organ bath fluid post-agonist-exposure. In dermal arteries, cinnamaldehyde- and capsaicin-induced relaxation remained unchanged after the aforementioned antagonists, while PregS-induced relaxation was significantly inhibited by isosakuranetin, L-NAME and MK-801. Furthermore, there was a significant increase in CGRP levels post-agonist-exposure. In our experimental model, TRPA1 and TRPV1 channels seem not to be involved in cinnamaldehyde- or capsaicin-induced relaxation, respectively, whereas TRPM3 channels contribute to PregS-induced relaxation, possibly via CGRP-independent mechanisms. Full article

Calcium-activated potassium channels (KCa) are important participants in calcium signaling pathways due to their ability to be activated by an increase in intracellular free calcium concentration. KCa channels are involved in the regulation of cellular processes in both normal and pathophysiological conditions, including oncotransformation. Previously, using patch-clamp, we registered the KCa currents in the plasma membrane of human chronic myeloid leukemia K562 cells, whose activity was controlled by local Ca²⁺ entry via mechanosensitive calcium-permeable channels. Here, we performed the molecular and functional identification of KCa channels and have uncovered their role in the proliferation, migration and invasion of K562 cells. Using a combined approach, we identified the functional activity of SK2, SK3 and IK channels in the plasma membrane of the cells. Selective SK and IK channel inhibitors, apamin and TRAM-34, respectively, reduced the proliferative, migratory and invasive capabilities of human myeloid leukemia cells. At the same time, the viability of K562 cells was not affected by KCa channel inhibitors. Ca²⁺ imaging showed that both SK and IK channel inhibitors affect Ca²⁺ entry and this could underlie the observed suppression of pathophysiological reactions of K562 cells. Our data imply that SK/IK channel inhibitors could be used to slow down the proliferation and spreading of chronic myeloid leukemia K562 cells that express functionally active KCa channels in the plasma membrane. Full article

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. Full article