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.

Apamin sensitive potassium current (I KAS), carried by the type 2 small conductance Ca(2+)-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

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.

Cholestatic liver disease is characterized by the progressive destruction of biliary epithelial cells (BECs) followed by fibrosis, cirrhosis and liver failure. Activated hepatic stellate cells (HSCs) and portal fibroblasts are the major cellular effectors of enhanced collagen deposition in biliary fibrosis. Apamin, an 18 amino acid peptide neurotoxin found in apitoxin (bee venom), is known to block Ca2+-activated K+ channels and prevent carbon tetrachloride-induced liver fibrosis. In the present study, we aimed to ascertain whether apamin inhibits biliary fibrosis and the proliferation of HSCs. Cholestatic liver fibrosis was established in mouse models with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Cellular assays were performed on HSC-T6 cells (rat immortalized HSCs). DDC feeding led to increased hepatic damage and proinflammtory cytokine levels. Notably, apamin treatment resulted in decreased liver injury and proinflammatory cytokine levels. Moreover, apamin suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice. In HSCs, apamin suppressed activation of HSCs by inhibiting the Smad signaling pathway. These data suggest that apamin may be a potential therapeutic target in cholestatic liver disease.

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-β1) 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.

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-1β, 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.

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.

Apamin, a peptide component of bee venom (BV), has anti-inflammatory properties. However, the molecular mechanisms by which apamin prevents atherosclerosis are not fully understood. We examined the effect of apamin on atherosclerotic mice. Atherosclerotic mice received intraperitoneal (ip) injections of lipopolysaccharide (LPS, 2 mg/kg) to induce atherosclerotic change and were fed an atherogenic diet for 12 weeks. Apamin (0.05 mg/kg) was administered by ip injection. LPS-induced THP-1-derived macrophage inflammation treated with apamin reduced expression of tumor necrosis factor (TNF)-α, vascular cell adhesion molecule (VCAM)-1, and intracellular cell adhesion molecule (ICAM)-1, as well as the nuclear factor kappa B (NF-κB) signaling pathway. Apamin decreased the formation of atherosclerotic lesions as assessed by hematoxylin and elastic staining. Treatment with apamin reduced lipids, Ca(2+) levels, and TNF-α in the serum from atherosclerotic mice. Further, apamin significantly attenuated expression of VCAM-1, ICAM-1, TGF-β1, and fibronectin in the descending aorta from atherosclerotic mice. These results indicate that apamin plays an important role in monocyte/macrophage inflammatory processing and may be of potential value for preventing atherosclerosis.

Bee venom (BV) has been used for the treatment of inflammatory diseases, such as rheumatoid arthritis, and for the relief of pain in traditional oriental medicine.

We previously reported that IKAS are heterogeneously upregulated in failing rabbit ventricles and play an important role in arrhythmogenesis. This study goal is to test the hypothesis that subtype 2 of the small-conductance Ca(2+) activated K(+) (SK2) channel and apamin-sensitive K(+) currents (IKAS) are upregulated in failing human ventricles.

1. It has been suggested that pituitary adenylate cyclase activating peptide (PACAP) may be involved in the non-adrenergic, non-cholinergic (NANC) inhibitory response of longitudinal muscle of rat distal colon. In this study, we have investigated the intracellular mechanism of PACAP-induced relaxation in this muscle. 2. PACAP induced an apamin-sensitive relaxation of the longitudinal muscle. The tyrosine kinase inhibitors genistein at 10 microM and tyrphostin 25 at 30 microM, but not the cyclic AMP-dependent protein kinase inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate at 30 microM significantly inhibited the PACAP-induced relaxation to 60% and 25% of control values, respectively. PACAP did not increase the cyclic AMP content of the muscle. 3. Tyrphostin 25 at 10 microM significantly inhibited the relaxation of longitudinal muscle induced by electrical field stimulation (EFS), to 50% of control values. Apamin at 1 microM, an antagonist of small conductance Ca2+-activated K+ channels, also inhibited the relaxation, to 42 % of control values. The inhibitory effects of tyrphostin 25 and apamin were not additive (44 % of control values). 4. PACAP induced an apamin-sensitive, slow hyperpolarization of the cell membrane of the muscle. Tyrphostin 25 at 3 microM inhibited this PACAP-induced hyperpolarization. Tyrphostin 25 at 10 microM and genistein at 10 microM inhibited the apamin-sensitive inhibitory junction potentials induced by a single pulse of EFS. 5. The PACAP-induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca2+ levels ([Ca2+]i). Tyrphostin 25 at 10 microM and apamin at 1 microM abolished these PACAP-induced responses. 6. From these findings it is suggested that the activation of tyrosine kinase is involved in PACAP-induced relaxation of longitudinal muscle from rat distal colon, 'upstream of' the activation of apamin-sensitive K+ channels.

SK2- and KV4.2-containing K+ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca2+ source that provides Ca2+-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca2+ entry through NMDARs, whereas KV4.2-containing channel availability is increased by Ca2+ entry through SNX-482 (SNX) sensitive CaV2.3 R-type Ca2+ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca2+ entry through R-type Ca2+ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca2+ channels and KV4.2-containing K+ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca2+ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from CaV2.3 null mice. The results show that in the absence of CaV2.3 channels, apamin application still boosted EPSPs. The boosting effect of CaV2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from CaV2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and KV4.2-containing channels to CaV2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.

Bee venom has recently been suggested to possess beneficial effects in the treatment of Parkinson disease (PD). For instance, it has been observed that bilateral acupoint stimulation of lower hind limbs with bee venom was protective in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In particular, a specific component of bee venom, apamin, has previously been shown to have protective effects on dopaminergic neurons in vitro. However, no information regarding a potential protective action of apamin in animal models of PD is available to date. The specific goals of the present study were to (i) establish that the protective effect of bee venom for dopaminergic neurons is not restricted to acupoint stimulation, but can also be observed using a more conventional mode of administration and to (ii) demonstrate that apamin can mimic the protective effects of a bee venom treatment on dopaminergic neurons. Using the chronic mouse model of MPTP/probenecid, we show that bee venom provides sustained protection in an animal model that mimics the chronic degenerative process of PD. Apamin, however, reproduced these protective effects only partially, suggesting that other components of bee venom enhance the protective action of the peptide.

Multiple sclerosis (MS) is a demyelinating disease that causes chronic inflammation in the central nervous system. The aim of this study was to investigate the effects of apamin administration on myelination process. MS was induced by feeding cuprizone pellets (0.2%) for 6 weeks (demyelination phase) followed by normal feeding for additional 2 weeks (remyelination phase). Briefly, C57BL/6 male mice were randomly divided into six groups. Group 1, received the regular food pellets. Group 2 contained two subgroups of 6 animals each (n = 2 × 6). First group received cuprizone for 6 weeks and the sacrificed while the second group after 6 weeks of cuprizone, received no treatment for additional 2 weeks. Group 3 (co-treatment group) was composed of two subgroups of 6 animals each (n = 2 × 6). Both subgroups received apamin (100 μg/kg) intraperitoneally twice a week for 6 weeks. First subgroup terminated at this time and the second subgroup was fed normal diet for two additional weeks. Group 4 (post-treatment, n = 6) received apamin (100 μg/kg) intraperitoneally twice a week for 2 weeks after cuprizone secession. Groups 5 and 6 (vehicle, n = 6 in each group) received phosphate buffered saline as the vehicle of apamin during demyelination and remyelination phase. At the end of each phase, mice were deeply anesthetized and perfused. Groups 5 and 6 (vehicle) received PBS as the vehicle during both phases. Mice were anesthetized, perfused with PBS through their heart, and their brains were removed. Brain sections stained with luxol fast blue and the images were analyzed. Apamin co-treatment significantly increased the myelin content as compared to the cuprizone group. Also, mild elevation in the myelinated areas was observed with apamin post-treatment in comparison with remyelination phase. Our results revealed that apamin prevents myelin destruction more significantly as compared to remyelination process. This observation explains the possible role of apamin in inhibiting the activation of the microglia cells than stimulation of the oligodendrocytic precursor cells.

Apamin is an integral part of bee venom, as a peptide component. It has long been known as a highly selective block Ca(2+)-activated K(+) (SK) channels. However, the cellular mechanism and anti-fibrotic effect of apamin in TGF-β1-induced hepatocytes have not been explored. In the present study, we investigated the anti-fibrosis or anti-EMT mechanism by examining the effect of apamin on TGF-β1-induced hepatocytes. AML12 cells were seeded at ∼60% confluence in complete growth medium. Twenty-four hours later, the cells were changed to serum free medium containing the indicated concentrations of apamin. After 30 min, the cells were treated with 2 ng/ml of TGF-β1 and co-cultured for 48 h. Also, we investigated the effects of apamin on the CCl4-induced liver fibrosis animal model. Treatment of AML12 cells with 2 ng/ml of TGF-β1 resulted in loss of E-cadherin protein at the cell-cell junctions and concomitant increased expression of vimentin. In addition, phosphorylation levels of ERK1/2, Akt, Smad2/3 and Smad4 were increased by TGF-β1 stimulation. However, cells treated concurrently with TGF-β1 and apamin retained high levels of localized expression of E-cadherin and showed no increase in vimentin. Specifically, treatment with 2 μg/ml of apamin almost completely blocked the phosphorylation of ERK1/2, Akt, Smad2/3 and Smad4 in AML12 cells. In addition, apamin exhibited prevention of pathological changes in the CCl4-injected animal models. These results demonstrate the potential of apamin for the prevention of EMT progression induced by TGF-β1 in vitro and CCl4-injected in vivo.

Neuroinflammation plays a vital role in neurodegenerative conditions. Microglia are a key component of the neuroinflammatory response. There is a growing interest in developing drugs to target microglia and thereby control neuroinflammatory processes. Apamin (APM) is a specifically selective antagonist of small conductance calcium-activated potassium (SK) channels. However, its effect on neuroinflammation is largely unknown. We examine the effects of APM on lipopolysaccharide (LPS)-stimulated BV2 and rat primary microglial cells. Regarding the molecular mechanism by which APM significantly inhibits proinflammatory cytokine production and microglial cell activation, we found that APM does so by reducing the expression of phosphorylated CaMKII and toll-like receptor (TLR4). In particular, APM potently suppressed the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/signal transducer and activator of transcription (STAT)3 and phosphorylated mitogen-activated protein kinases (MAPK)-extracellular signal-regulated kinase (ERK). In addition, the correlation of NF-κB/STAT3 and MAPK-ERK in the neuroinflammatory response was verified through inhibitors. The literature and our findings suggest that APM is a promising candidate for an anti-neuroinflammatory agent and can potentially be used for the prevention and treatment of various neurological disorders.

Apamin is commonly used as a small-conductance Ca2+-activated K+ (SK) current inhibitor. However, the specificity of apamin in cardiac tissues remains unclear.

Certain anticancer agents selectively target the nucleus of cancer cells. One such drug is 2-methoxyestradiol (2ME), which is used for treating lung cancer. To improve the therapeutic effectiveness of these agents, many new methods have been devised. 2ME was entrapped into the core of hydrophobic invasomes (INVA) covered with Phospholipon 90G and apamin (APA). The Box-Behnken statistical design was implemented to enhance the composition. Using Design-Expert software (Stat-Ease Inc., Minneapolis, MN), the INVA component quantities were optimized to obtain spherical particles with the smallest size, that is, a diameter of 167.8 nm. 2ME-INVA-APA significantly inhibited A549 cells and exhibited IC50 of 1.15 ± 0.04 µg/mL, which is lower than raw 2ME (IC50 5.6 ± 0.2 µg/mL). Post 2ME-INVA-APA administration, a significant rise in cell death and necrosis was seen among the A549 cells compared to those treated with plain formula or 2ME alone. This effect was indicated by increased Bax expression and reduced Bcl-2 expression, as well as mitochondrial membrane potential loss. Moreover, the cell cycle analysis showed that 2ME-INVA-APA arrests the G2-M phase of the A549 cells. Additionally, it was observed that the micellar formulation of the drug increased the cell count in pre-G1, thereby exhibiting phenomenal apoptotic potential. Furthermore, it up-regulates caspase-9 and p53 and downregulates TNF-α and NF-κβ. Collectively, these findings showed that our optimized 2ME-INVA-APA could easily seep through the cell membrane and induce apoptosis in relatively low doses.

Sudden and complete unilateral loss of peripheral vestibular inputs evokes characteristic vestibular syndrome comprised of posturo-locomotor, oculomotor, vegetative and cognitive symptoms. Subsequently to the vestibular insult, a neurophysiological process called central vestibular compensation promotes the progressive restoration of the posture and balance. The modulation of the excitability of vestibular secondary neurons has been demonstrated to be a key process of this mechanism. However, the molecular mechanisms that support this modulatory process have thus far not been fully identified. The present study used a combination of a radio-labeled apamin binding experiment and a functional assessment of the vestibular function to demonstrate that unilateral vestibular neurectomy (UVN) induces both ipsi- and contralateral up-regulation of the apamin-sensitive calcium-activated small conductance K+ (SK) channels, within the first days following the insult. We also demonstrate that apamin administration during the acute phase of the vestibular syndrome significantly reduces both the posturo-locomotor and vestibulo-ocular deficits induced by the UVN. This is illustrated by the reduction of both the spontaneous nystagmus and the static and dynamic balance unsteadiness. These data suggest that the regulation of SK channel expression may be part of the vestibular compensation process. It is also indicated that the pharmacological modulation of SK channels may be a potential way to alleviate the vestibular syndrome.

Sepsis is the major cause of acute kidney injury (AKI) in severely ill patients, but only limited therapeutic options are available. During sepsis, lipopolysaccharide (LPS), an endotoxin derived from bacteria, activates signaling cascades involved in inflammatory responses and tissue injury. Apamin is a component of bee venom and has been shown to exert antioxidative, antiapoptotic, and anti-inflammatory activities. However, the effect of apamin on LPS-induced AKI has not been elucidated. Here, we show that apamin treatment significantly ameliorated renal dysfunction and histological injury, especially tubular injury, in LPS-injected mice. Apamin also suppressed LPS-induced oxidative stress through modulating the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 and heme oxygenase-1. Moreover, tubular cell apoptosis with caspase-3 activation in LPS-injected mice was significantly attenuated by apamin. Apamin also inhibited cytokine production and immune cell accumulation, suppressed toll-like receptor 4 pathway, and downregulated vascular adhesion molecules. Taken together, these results suggest that apamin ameliorates LPS-induced renal injury through inhibiting oxidative stress, apoptosis of tubular epithelial cells, and inflammation. Apamin might be a potential therapeutic option for septic AKI.