Apamin Treatment Research Articles

Lack of UBE3A-Mediated Regulation of Synaptic SK2 Channels Contributes to Learning and Memory Impairment in the Female Mouse Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe developmental delay, motor impairment, language and cognition deficits, and often with increased seizure activity. AS is caused by deficiency of UBE3A, which is both an E3 ligase and a cofactor for transcriptional regulation. We previously showed that the small conductance potassium channel protein SK2 is a UBE3A substrate, and that increased synaptic SK2 levels contribute to impairments in synaptic plasticity and fear-conditioning memory, as inhibition of SK2 channels significantly improved both synaptic plasticity and fear memory in male AS mice. In the present study, we investigated UBE3a-mediated regulation of synaptic plasticity and fear-conditioning in female AS mice. Results from both western blot and immunofluorescence analyses showed that synaptic SK2 levels were significantly increased in hippocampus of female AS mice, as compared to wild-type (WT) littermates. Like in male AS mice, long-term potentiation (LTP) was significantly reduced while long-term depression (LTD) was enhanced at hippocampal CA3-CA1 synapses of female AS mice, as compared to female WT mice. Both alterations were significantly reduced by treatment with the SK2 inhibitor, apamin. The shunting effect of SK2 channels on NMDA receptor was significantly larger in female AS mice as compared to female WT mice. Female AS mice also showed impairment in both contextual and tone memory recall, and this impairment was significantly reduced by apamin treatment. Our results indicate that like male AS mice, female AS mice showed significant impairment in both synaptic plasticity and fear-conditioning memory due to increased levels of synaptic SK2 channels. Any therapeutic strategy to reduce SK2-mediated inhibition of NMDAR should be beneficial to both male and female patients.

Apamin inhibits renal fibrosis via suppressing TGF-β1 and STAT3 signaling in vivo and in vitro.

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-β1 (TGF-β1) 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-β1-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-1β, and IL-6 by suppressing the TGF-β1-canonical and non-canonical signaling pathways. This study showed that apamin inhibits UUO-induced renal fibrosis in vivo and TGF-β1-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-β1/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-β1 canonical and non-canonical signaling.

Antioxidative, Antiapoptotic, and Anti-Inflammatory Effects of Apamin in a Murine Model of Lipopolysaccharide-Induced Acute Kidney Injury.

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.

Apamin from bee venom suppresses inflammation in a murine model of gouty arthritis

Ethnopharmacological relevanceBee 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. Aim of studyThe aim of this study was to determine the anti-inflammatory effect of BV on monosodium urate (MSU)-induced gouty arthritis in a mouse model. Materials and methodsTo develop a mouse model of acute gouty arthritis, 4 mg 50 μL−1 of MSU crystal suspension was injected intradermally into the right paw. After MSU crystal injection, we evaluated inflammatory cytokine production in mice of the BV-treated (0.5 and 1 mg kg−1 body weight) and apamin (APM)-treated (0.5 and 1 mg kg−1 body weight) groups. The positive control group was administered a colchicine (1 mg kg−1 body weight) injection with MSU crystals. ResultsBV and APM treatment suppressed inflammatory paw edema in MSU-administered mice. It also exerted anti-inflammatory effects in mice with gouty arthritis by inhibiting proinflammatory cytokine production and inflammasome formation. Interestingly, MSU crystal formation was decreased by BV and APM treatment. ConclusionsThese results suggest that the APM from BV might be useful for the treatment of gouty arthritis due to its anti-inflammatory activities.

Apamin treatment accelerates equilibrium recovery and gaze stabilization in unilateral vestibular neurectomized cats: Cellular and behavioral aspects

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.

Apamin suppresses biliary fibrosis and activation of hepatic stellate cells.

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.

SK channel blockade reverses cognitive and motor deficits induced by nigrostriatal dopamine lesions in rats

Parkinson's disease has traditionally been viewed as a motor disorder caused by the loss of dopamine (DA) neurons. However, emotional and cognitive syndromes can precede the onset of the motor deficits and provide an opportunity for therapeutic intervention. Potassium channels have recently emerged as potential new targets in the treatment of Parkinson's disease. The selective blockade of small conductance calcium-activated K+ channels (SK channels) by apamin is known to increase burst firing in midbrain DA neurons and therefore DA release. We thus investigated the effects of systemic administration of apamin on the motor, cognitive deficits and anxiety present after bilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesions in rats. Apamin administration (0.1 or 0.3mg/kg i.p.) counteracted the depression, anxiety-like behaviors evaluated on sucrose consumption and in the elevated plus maze, social recognition and spatial memory deficits produced by partial 6-OHDA lesions. Apamin also reduced asymmetric motor deficits on circling behavior and postural adjustments in the unilateral extensive 6-OHDA model. The partial 6-OHDA lesions (56% striatal DA depletion) produced 20% decrease of iodinated apamin binding sites in the substantia nigra pars compacta in correlation with the loss of tyrosine hydroxylase positive cells, without modifying apamin binding in brain regions receiving DAergic innervation. Striatal extracellular levels of DA, not detectable after 6-OHDA lesions, were enhanced by apamin treatment as measured by in vivo microdialysis. These results indicate that blocking SK channels may reinstate minimal DA activity in the striatum to alleviate the non-motor symptoms induced by partial striatal DA lesions.

Apamin Attenuated Cerulein-Induced Acute Pancreatitis by Inhibition of JNK Pathway in Mice

We have previously reported that bee venom (BV) has a protective role against acute pancreatitis (AP). However, the effects of apamin, the major compound of BV, on AP have not been determined. The aim of this study was to evaluate the effects of apamin on cerulein-induced AP. AP was induced via intraperitoneal injection of supramaximal concentrations of the stable cholecystokinin analogue cerulein (50 μg/kg) every hour for 6 times. In the apamin treatment group, apamin was administered subcutaneously (10, 50, or 100 μg/kg) at both 18 and 1 h before the first cerulein injection. The mice were sacrificed at 6 h after the final cerulein injection. Blood samples were obtained to determine serum amylase and lipase levels, as well as cytokine production. The pancreas and lung were rapidly removed for morphologic and histological examination, myeloperoxidase (MPO) assay, and real-time reverse transcription-polymerase chain reaction. Furthermore, we isolated the pancreatic acinar cells to specify the role of apamin in AP. Pre-treatment with apamin inhibited histological damage, pancreatic weight/body weight ratio, serum level of amylase and lipase, MPO activity, and cytokine production. In addition, apamin treatment significantly inhibited cerulein-induced pancreatic acinar cell death. Furthermore, apamin treatment inhibited the cerulein-induced activation of c-Jun NH2-terminal kinases (JNK). These results could suggest that apamin could protect against AP by inhibition of JNK activation.

Apamin inhibits THP-1-derived macrophage apoptosis via mitochondria-related apoptotic pathway

The development of atherosclerotic lesions is mainly due to macrophage death. The oxidative stresses of monocytes/macrophages play a vital role in the initiation and amplification of atherosclerosis. Apamin, a component of bee venom, exerts an anti-inflammatory effect, and selectively inhibits the Ca2+-activated K+ channels. The mechanisms involved in the inhibition of macrophage apoptosis have been fully elucidated.We induced oxidized low-density lipoprotein (oxLDL) in THP-1-derived macrophage and studied the effect of apamin on intercellular lipid levels, mitochondria-related apoptotic pathway and numbers of apoptotic cells.Oil-red O staining indicates that the inhibition of apamin in the condition significantly prevents intracellular lipid deposition. Treatment with apamin significantly decreased the apoptotic macrophages by decreasing the expression of pro-apoptotic genes Bax, caspase-3 and PARP protein levels, as well as through increasing expression of anti-apoptotic genes Bcl-2 and Bcl-xL protein levels in the absence and presence of oxLDL. In vivo, with apamin treatment reduced apoptotic cells death by TUNEL staining.These results indicate that apamin plays an important role in monocyte/macrophage apoptotic processing, which may provide a potential drug for preventing atherosclerosis.

Differential Effects of K<sup>+</sup> Channel Blockers on Phasic Contractility of Transverse and Longitudinal Rat Detrusor Strips

Spontaneous phasic contractions of detrusor smooth muscle are pivotal to the normal bladder filling process. The role of K+ channels in mediating phasic contractions has been investigated on different occasions, but only in detrusor strips isolated longitudinally. In this study, the effects of individual K+ blockers were examined in both transverse and longitudinal detrusor strips. Detrusor strips were isolated transversely and longitudinally from young adult rat bladders. Tension before and after the introduction of K+channel blockers was measured using a myograph. Phasic activity was determined by calculating the integral of tension fluctuations. Phasic activity of transverse strips was increased under tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and iberiotoxin (IbTx) treatments. Longitudinal phasic activity was increased under charybdotoxin (ChTx) treatment. Neither glibenclamide (Glib) nor apamin treatment elicited any significant effect in both transverse and longitudinal phasic activity. The results indicated that phasic activity was mediated differently depending on the contractile direction. Data from this study reiterate that in addition to the conventional longitudinal direction, the transverse direction also presents significance when examining the contractility of a sac-like organ like the bladder.

Hypertension Induced by Angiotensin II and a High Salt Diet Involves Reduced SK Current and Increased Excitability of RVLM Projecting PVN Neurons

Although evidence indicates that activation of presympathetic paraventricular nucleus (PVN) neurons contributes to the pathogenesis of salt-sensitive hypertension, the underlying cellular mechanisms are not fully understood. Recent evidence indicates that small conductance Ca(2+)-activated K(+) (SK) channels play a significant role in regulating the excitability of a key group of sympathetic regulatory PVN neurons, those with axonal projections to the rostral ventrolateral medulla (RVLM; i.e., PVN-RVLM neurons). In the present study, rats consuming a high salt (2% NaCl) diet were made hypertensive by systemic infusion of angiotensin II (AngII), and whole cell patch-clamp recordings were made in brain slice from retrogradely labeled PVN-RVLM neurons. To determine if the amplitude of SK current was altered in neurons from hypertensive rats, voltage-clamp recordings were performed to isolate SK current. Results indicate that SK current amplitude (P < 0.05) and density (P < 0.01) were significantly smaller in the hypertensive group. To investigate the impact of this on intrinsic excitability, current-clamp recordings were performed in separate groups of PVN-RVLM neurons. Results indicate that the frequency of spikes evoked by current injection was significantly higher in the hypertensive group (P < 0.05-0.01). Whereas bath application of the SK channel blocker apamin significantly increased discharge of neurons from normotensive rats (P < 0.05-0.01), no effect was observed in the hypertensive group. In response to ramp current injections, subthreshold depolarizing input resistance was greater in the hypertensive group compared with the normotensive group (P < 0.05). Blockade of SK channels increased depolarizing input resistance in normotensive controls (P < 0.05) but had no effect in the hypertensive group. On termination of current pulses, a medium afterhyperpolarization potential (mAHP) was observed in most neurons of the normotensive group. In the hypertensive group, the mAHP was either small or absent. In the latter case, an afterdepolarization potential (ADP) was observed that was unaffected by apamin. Apamin treatment in the normotensive group blocked the mAHP and revealed an ADP resembling that seen in the hypertensive group. We conclude that diminished SK current likely underlies the absence of mAHPs in PVN-RVLM neurons from hypertensive rats. Both the ADP and greater depolarizing input resistance likely contribute to increased excitability of PVN-RVLM neurons from rats with AngII-Salt hypertension.

CRITICAL ROLE FOR SMALL AND LARGE CONDUCTANCE CALCIUM-DEPENDENT POTASSIUM CHANNELS IN ENDOTOXEMIA AND TNF TOXICITY

Sepsis-associated vasodilation and shock are centrally orchestrated by NO. Nevertheless, inhibition of NO synthesis may not be the target of choice for the treatment of septic shock because it increases morbidity and mortality. Potential other therapeutic targets include soluble guanulate cyclase (sGC) and K+ channels. In this study, we investigated the protective effect of the sGC inhibitor methylene blue (MB) and various K+ channel inhibitors on LPS- and TNF-induced mortality in mice. In TNF-induced shock, the importance of SK channels was underscored by the ability of a single treatment with the small-conductance calcium-activated SK channel inhibitor apamin to provide significant protection. The only other K+ channel inhibitor that can add survival benefit to the apamin treatment was iberiotoxin, stressing the importance of large-conductance calcium-activated BK channels as well. Although MB can protect against TNF-induced shock and mortality, it cannot prevent LPS-induced mortality. Treatment with the nonspecific K+ channel inhibitor tetraethylammonium or with inhibitors specific for adenosine triphosphate (ATP)-sensitive KATP channels (glibenclamide), BK channels (iberiotoxin), or SK channels (apamin) could not protect either. However, when we combined MB treatment with a single dose of apamin and iberiotoxin, mice were completely protected against LPS-induced death for at least 2 days. In conclusion, the protective effect of MB in combination with apamin and iberiotoxin indicates an important role for SK and BK channels, rather than KATP channels, during endotoxemia. Our results point to SK and BK channels as potential targets in septic shock treatment to be modulated preferentially together with sGC.

Identification of SK3 channel as a new mediator of breast cancer cell migration

Potassium channels have been involved in epithelial tumorigenesis but the role of small-conductance Ca(2+)-activated K(+) channels is unknown. We report here that small-conductance Ca(2+)-activated K(+) channels are expressed in a highly metastasizing mammary cancer cell line, MDA-MB-435s. Patch-clamp recordings showed typical small-conductance Ca(2+)-activated K(+) channel-mediated currents sensitive to apamin, 4-aminopyridine, and tetraethylammonium. Moreover, the cells displayed a high intracellular calcium concentration, which was decreased after 24 hours of apamin treatment. By regulating membrane potential and intracellular calcium concentration, these channels were involved in MDA-MB-435s cell migration, but not in proliferation. Only SK3 protein expression was observed in these cells in contrast to SK2, which was expressed both in cancer and noncancer cell lines. Whereas small interfering RNA directed against SK3 almost totally abolished MDA-MB-435s cell migration, transient expression of SK3 increased migration of the SK3-deficient cell lines, MCF-7 and 184A1. SK3 channel was solely expressed in tumor breast biopsies and not in nontumor breast tissues. Thus, SK3 protein channel seems to be a new mediator of breast cancer cell migration and represents a potential target for a new class of anticancer agents.

The different mechanisms of action of nicorandil and adenosine triphosphate on potassium channels of circular smooth muscle of the guinea-pig small intestine

Nicorandil (10 mumol/l-0.3 mmol/l) and ATP (1 mumol/l - 0.1 mmol/l) hyperpolarized the membrane of circular smooth muscle of the guinea-pig small intestine and increased conductance of the membrane probably to K ions as estimated by the effect on the current-voltage relationship. In the presence of a maximally hyperpolarizing concentration of nicorandil (0.1 mmol/l), ATP produced a further hyperpolarization of 5 mV. The ATP-induced but not the nicorandil-induced hyperpolarization required the presence of Ca in the medium, and the ATP-induced hyperpolarization was blocked by apamin treatment (1 nmol/l) or by MnCl2 (1.3 mmol/l). On the other hand, both hyperpolarization responses were blocked by the local anaesthetics procaine (0.1 - 1 mmol/l), lidocaine (0.1 - 1 mmol/l) or cocaine (0.3 - 1 mmol/l), with different potencies. Field stimulation of smooth muscle of the small intestine produced inhibitory junction potentials (i.j.p.s) and these were inhibited by apamin (10 nmol/l - 100 nmol/l). In the presence of ATP, the amplitude of the i.j.p.s was markedly reduced, but in the presence of nicorandil the amplitude was only slightly reduced, consistent with the same increase in ionic conductance and hyperpolarization of the membrane. These results indicate that ATP and nicorandil hyperpolarize the membrane by activating different K-channels, i.e. Ca dependent and Ca insensitive K channels, respectively. As assessed from the effects of local anaesthetics and the membrane properties, the circular muscle may also possess other K channels different from the ATP and nicorandil sensitive K channels.

Evidence against vasoactive intestinal polypeptide being the non-adrenergic, non-cholinergic inhibitory transmitter released from nerves supplying the smooth muscle of the guinea-pig taenia coli

Vasoactive intestinal polypeptide (VIP), located within intrinsic neurones of the guinea-pig taenia coli, has been proposed as a neurotransmitter which may mediate certain non-adrenergic, non-aholinerfic effects within the gastrointestinal tract. The relaxation of the taenia coli produced by exogenous VIP has a longer latency and time to maximum than the relaxation which is obtained during electrical stimulation of non-adrenergic, non-cholinergic nerves. The responses to exogenous VIP (0.03– μM) were abolished by the proteolytic enzyme α-chymotrypsin (1 U/ml), whereas there were no statistically significant changes in the inhibitory responses to intramural nerve stimulation (0.1–5 Hz) or exogenous ATP (0.03–100 μM), which closely mimics the nerve-mediated response. Apamin is a neurotoxin which prevents the increase in K + conductance associated with the inhibitory junction potential produced by stimulation of the non-adrenergic, non-cholinergic inhibitory nerves. In the presence of apamin (0.005–1 μM) the responses to intramural nerve stimulation and exogenous ATP were significantly antagonised, and often converted to contractile responses. The VIP-induced relaxations during apamin treatment were not significantly decreased. These results suggests that VIP is unlikely to be the transmitter released from the non-adrenergic, non-cholinergic nerves of the guinea-pig taenia coli, and are consistent with the view that these nerves are purinergic in nature.