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1.
ACS Chem Neurosci ; 14(18): 3409-3417, 2023 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-37647501

RESUMO

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.


Assuntos
Disfunção Cognitiva , Doenças Neuroinflamatórias , Animais , Camundongos , Camundongos Endogâmicos C57BL , Apamina , Sevoflurano , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Cognição , Hipocampo
2.
Life Sci ; 330: 121942, 2023 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-37451399

RESUMO

AIMS: Although absorbed NaCl increases intestinal blood flow to facilitate absorption and transportation, it is unclear if it can directly mediate mesenteric arterial relaxation. We aimed to investigate and test our hypothesis that Cl- induces mesenteric arterial vasorelaxation via endothelium-dependent hyperpolarization (EDH). MAIN METHODS: We used wire myograph to study NaCl-induced vasorelaxation of mesenteric arteries isolated from mice. Cl-, Ca2+ and K+ imaging was performed in human vascular endothelial cells pre-treated with pharmacological agents. KEY FINDINGS: The Cl- concentration-dependently induced vasorelaxation of mesenteric arteries likely through EDH. The Cl--induced vasorelaxation was attenuated in TRPV4 KO mice and inhibited by selective blockers of Na+-K+-2Cl- cotransporter 1 (NKCC1) (bumetanide, 10 µM), transient receptor potential vanilloid 4 (TRPV4) (RN-1734, 40 µM), and small conductance Ca2+-activated K+ channels (SKCa) (apamin, 3 µM)/ intermediate conductance Ca2+-activated K+ channels (IKCa) (TRAM-34, 10 µM) and myoendothelial gap junction (18α-glycyrrhetinic acid, 10 µM), but enhanced by a selective activator of IKCa/SKCa (SKA-31, 0.3 µM). Cl- decreased intracellular K+ concentrations in endothelial cells, which was reversed by apamin (200 nM) plus TRAM-34 (500 nM). Extracellular Cl- raised intracellular Cl- concentrations in endothelial cells, which was attenuated by bumetanide (10 µM). Finally, Cl- induced a transient Ca2+ signaling via TRPV4 in endothelial cells, which became sustained when the Ca2+ exit mode of Na+-Ca2+ exchanger (NCX) was blocked. SIGNIFICANCE: Cl- induces a pure EDH-mediated vasorelaxation of mesenteric arteries through activation of endothelial NKCC1/TRPV4/NCX axis. We have provided a novel insight into the role of Cl--induced vasorelaxation via EDH mechanism.


Assuntos
Canais de Cátion TRPV , Vasodilatação , Camundongos , Humanos , Animais , Vasodilatação/fisiologia , Células Endoteliais , Bumetanida , Cloreto de Sódio , Apamina , Artérias Mesentéricas , Endotélio Vascular
3.
PLoS Comput Biol ; 19(4): e1010993, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37068087

RESUMO

Dorsal horn of the spinal cord is an important crossroad of pain neuraxis, especially for the neuronal plasticity mechanisms that can lead to chronic pain states. Windup is a well-known spinal pain facilitation process initially described several decades ago, but its exact mechanism is still not fully understood. Here, we combine both ex vivo and in vivo electrophysiological recordings of rat spinal neurons with computational modeling to demonstrate a role for ASIC1a-containing channels in the windup process. Spinal application of the ASIC1a inhibitory venom peptides mambalgin-1 and psalmotoxin-1 (PcTx1) significantly reduces the ability of deep wide dynamic range (WDR) neurons to develop windup in vivo. All deep WDR-like neurons recorded from spinal slices exhibit an ASIC current with biophysical and pharmacological characteristics consistent with functional expression of ASIC1a homomeric channels. A computational model of WDR neuron supplemented with different ASIC1a channel parameters accurately reproduces the experimental data, further supporting a positive contribution of these channels to windup. It also predicts a calcium-dependent windup decrease for elevated ASIC conductances, a phenomenon that was experimentally validated using the Texas coral snake ASIC-activating toxin (MitTx) and calcium-activated potassium channel inhibitory peptides (apamin and iberiotoxin). This study supports a dual contribution to windup of calcium permeable ASIC1a channels in deep laminae projecting neurons, promoting it upon moderate channel activity, but ultimately leading to calcium-dependent windup inhibition associated to potassium channels when activity increases.


Assuntos
Cálcio , Dor , Animais , Ratos , Cálcio/metabolismo , Simulação por Computador , Neurônios/fisiologia , Peptídeos , Apamina/metabolismo
4.
Circ Res ; 132(9): e116-e133, 2023 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-36927079

RESUMO

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.


Assuntos
Fibrilação Atrial , Animais , Humanos , Fibrilação Atrial/metabolismo , Apamina/metabolismo , Apamina/farmacologia , Primaquina/metabolismo , Primaquina/farmacologia , Calmodulina/metabolismo , Átrios do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Antiarrítmicos/uso terapêutico , Potenciais de Ação/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
5.
Biophys J ; 122(7): 1143-1157, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-36760125

RESUMO

Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τc,vl = 11.8 ± 7.1 s, and AP14145: τc,vl = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τc,s = 3.50 ± 0.81 ms, and τc,i = 32.0 ± 6.76 ms versus control: τc,s = 1.59 ± 0.19 ms, and τc,i = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τc,s = 0.75 ± 0.10 ms and τc,i = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: Ahigh = 0.76 ± 0.03 pA, and Alow = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca2+-dependent channel activation in vivo.


Assuntos
Canais de Potássio , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Apamina/farmacologia , Acetamidas , Cinética , Cálcio/metabolismo
6.
J Physiol ; 601(1): 51-67, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36426548

RESUMO

At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We tested the hypothesis that in single rabbit ventricular myocytes pharmacological modulation of SK channels plays a causative role for the development of pacing-induced CaT and AP duration (APD) alternans. SK channel blockers (apamin, UCL1684) had only a minor effect on AP repolarization. However, SK channel activation by NS309 resulted in significant APD shortening, demonstrating that functional SK channels are well expressed in ventricular myocytes. The effects of NS309 were prevented or reversed by apamin and UCL1684, indicating that NS309 acted on SK channels. SK channel activation abolished or reduced the degree of pacing-induced CaT and APD alternans. Inhibition of KV 7.1 (with HMR1556) and KV 11.1 (with E4031) channels was used to mimic conditions of long QT syndromes type-1 and type-2, respectively. Both HMR1556 and E4031 enhanced CaT alternans that was prevented by SK channel activation. In AP voltage-clamped cells the SK channel activator had no effect on CaT alternans, confirming that suppression of CaT alternans was caused by APD shortening. APD shortening contributed to protection from alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest that SK activation could be a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy for patients with long QT syndrome. KEY POINTS: At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and intracellular Ca2+ release amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We investigated whether pharmacological modulation of SK channels affects the development of cardiac alternans in normal ventricular cells and in cells with drug-induced long QT syndrome (LQTS). While SK channel blockers have only a minor effect on AP morphology, their activation leads to AP shortening and abolishes or reduces the degree of pacing-induced Ca2+ and AP alternans. AP shortening contributed to protection against alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert the development of alternans with important ramifications for arrhythmia prevention for patients with LQTS.


Assuntos
Arritmias Cardíacas , Síndrome do QT Longo , Animais , Coelhos , Potenciais de Ação/fisiologia , Apamina/farmacologia , Miócitos Cardíacos/fisiologia , Doença do Sistema de Condução Cardíaco
7.
J Biol Chem ; 299(1): 102783, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36502918

RESUMO

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Baixa , Humanos , Apamina/farmacologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Baixa/química , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
8.
Europace ; 26(1)2023 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-38195705

RESUMO

AIMS: Metabolic syndrome (MetS) is associated with arrhythmias and cardiovascular mortality. Arrhythmogenesis in MetS results from atrial structural and electrical remodelling. The small-conductance Ca2+-activated K+ (SK) currents modulate atrial repolarization and may influence atrial arrhythmogenicity. This study investigated the regulation of SK current perturbed by a high-fat diet (HFD) to mimic MetS. METHODS AND RESULTS: Thirty mice were divided into two groups that were fed with normal chow (CTL) and HFD for 4 months. Electrocardiography and echocardiography were used to detect cardiac electrical and structure remodelling. Atrial action potential duration (APD) and calcium transient duration (CaTD) were measured by optical mapping of Langendorff-perfused mice hearts. Atrial fibrillation (AF) inducibility and duration were assessed by burst pacing. Whole-cell patch clamp was performed in primarily isolated atrial myocytes for SK current density. The SK current density is higher in atrial myocytes from HFD than in CTL mice (P ≤ 0.037). The RNA and protein expression of SK channels are increased in HFD mice (P ≤ 0.041 and P ≤ 0.011, respectively). Action potential duration is shortened in HFD compared with CTL (P ≤ 0.015). The shortening of the atrial APD in HFD is reversed by the application of 100 nM apamin (P ≤ 0.043). Compared with CTL, CaTD is greater in HFD atria (P ≤ 0.029). Calcium transient decay (Tau) is significantly higher in HFD than in CTL (P = 0.001). Both APD and CaTD alternans thresholds were higher in HFD (P ≤ 0.043), along with higher inducibility and longer duration of AF in HFD (P ≤ 0.023). CONCLUSION: Up-regulation of apamin-sensitive SK currents plays a partial role in the atrial arrhythmogenicity of HFD mice.


Assuntos
Fibrilação Atrial , Cálcio , Camundongos , Animais , Cálcio/metabolismo , Potássio/metabolismo , Apamina/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Potenciais de Ação/fisiologia , Miócitos Cardíacos/metabolismo
9.
Sci Rep ; 12(1): 22168, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36550366

RESUMO

Most species of bee are capable of delivering a defensive sting which is often painful. A solitary lifestyle is the ancestral state of bees and most extant species are solitary, but information on bee venoms comes predominantly from studies on eusocial species. In this study we investigated the venom composition of the Australian great carpenter bee, Xylocopa aruana Ritsema, 1876. We show that the venom is relatively simple, composed mainly of one small amphipathic peptide (XYTX1-Xa1a), with lesser amounts of an apamin homologue (XYTX2-Xa2a) and a venom phospholipase-A2 (PLA2). XYTX1-Xa1a is homologous to, and shares a similar mode-of-action to melittin and the bombilitins, the major components of the venoms of the eusocial Apis mellifera (Western honeybee) and Bombus spp. (bumblebee), respectively. XYTX1-Xa1a and melittin directly activate mammalian sensory neurons and cause spontaneous pain behaviours in vivo, effects which are potentiated in the presence of venom PLA2. The apamin-like peptide XYTX2-Xa2a was a relatively weak blocker of small conductance calcium-activated potassium (KCa) channels and, like A. mellifera apamin and mast cell-degranulating peptide, did not contribute to pain behaviours in mice. While the composition and mode-of-action of the venom of X. aruana are similar to that of A. mellifera, the greater potency, on mammalian sensory neurons, of the major pain-causing component in A. mellifera venom may represent an adaptation to the distinct defensive pressures on eusocial Apidae.


Assuntos
Venenos de Abelha , Toxinas Biológicas , Abelhas , Camundongos , Animais , Meliteno , Apamina , Austrália , Venenos de Abelha/química , Fosfolipases A2 , Peptídeos , Dor/induzido quimicamente , Mamíferos
10.
Int J Mol Sci ; 23(23)2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36499581

RESUMO

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.


Assuntos
Neuroblastoma , Fármacos Neuroprotetores , Síndromes Neurotóxicas , Doença de Parkinson , Humanos , Ratos , Animais , Cálcio/metabolismo , Apamina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fármacos Neuroprotetores/farmacologia , Neuroblastoma/metabolismo , 1-Metil-4-fenilpiridínio/toxicidade , Neurônios Dopaminérgicos/metabolismo , Transdução de Sinais , Estresse Oxidativo , Doença de Parkinson/metabolismo , NF-kappa B/metabolismo , Síndromes Neurotóxicas/patologia , Apoptose , Linhagem Celular Tumoral
11.
Neural Plast ; 2022: 3923384, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36237484

RESUMO

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.


Assuntos
Síndrome de Angelman , Síndrome de Angelman/metabolismo , Animais , Apamina , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Masculino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/farmacologia
12.
Sci Rep ; 12(1): 16746, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36202914

RESUMO

Protease-activated receptor-1 (PAR1) is highly expressed in murine colonic smooth muscles. Responses to PAR1 activation are complex and result from responses in multiple cell types. We investigated whether PAR1 responses are altered in inflamed colon induced by dextran sodium sulfate (DSS)-treatment. Colitis was induced in C57BL/6 mice by administration of 3% DSS in drinking water for 7 days. Measurements of isometric force, transmembrane potentials from impaled smooth muscle cells, quantitative PCR and Western blots were performed. Thrombin, an activator of PAR1, caused transient hyperpolarization and relaxation of untreated colons, but these responses decreased in DSS-treated colons. Apamin caused depolarization and increased contractions of muscles from untreated mice. This response was decreased in DSS-treated colons. Expression of Kcnn3 and Pdgfra also decreased in DSS-treated muscles. A second phase of thrombin responses is depolarization and increased contractions in untreated muscles. However, thrombin did cause depolarization in DSS-treated colon, yet it increased colonic contractions. The latter effect was associated with enhanced expression of MYPT1 and CPI-17. The propagation velocity and frequency of colonic migrating motor complexes in DSS-treated colon was significantly higher compared to control colons. In summary, DSS treatment causes loss of transient relaxations due to downregulation of SK3 channels in PDGFRα+ cells and may increase contractile responses due to increased Ca2+ sensitization of smooth muscle cells via PAR1 activation.


Assuntos
Colite , Água Potável , Animais , Apamina/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colo/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Receptor PAR-1/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Sulfatos , Trombina/metabolismo
13.
Am J Physiol Heart Circ Physiol ; 323(5): H869-H878, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36149772

RESUMO

Atrial fibrillation (AF) is associated with electrical remodeling processes that promote a substrate for the maintenance of AF. Although the small-conductance Ca2+-activated K+ (SK) channel is a key factor in atrial electrical remodeling, the mechanism of its activation remains unclear. Regional nitric oxide (NO) production by neuronal nitric oxide synthase (nNOS) is involved in atrial electrical remodeling. In this study, atrial tachyarrhythmia (ATA) induction and optical mapping were performed on perfused rat hearts. nNOS is pharmacologically inhibited by S-methylthiocitrulline (SMTC). The influence of the SK channel was examined using a specific channel inhibitor, apamin (APA). Parameters such as action potential duration (APD), conduction velocity, and calcium transient (CaT) were evaluated using voltage and calcium optical mapping. The dominant frequency was examined in the analysis of AF dynamics. SMTC (100 nM) increased the inducibility of ATA and apamin (100 nM) mitigated nNOS inhibition-induced arrhythmogenicity. SMTC caused abbreviations and enhanced the spatial dispersion of APD, which was reversed by apamin. By contrast, conduction velocity and other parameters associated with CaT were not affected by SMTC or apamin administration. Apamin reduced the frequency of SMTC-induced ATA. In summary, nNOS inhibition abbreviates APD by modifying the SK channels. A specific SK channel blocker, apamin, mitigated APD abbreviation without alteration of CaT, implying an underlying mechanism of posttranslational modification of SK channels.NEW & NOTEWORTHY We demonstrated that pharmacological nNOS inhibition increased the atrial arrhythmia inducibility and a specific small-conductance Ca2+-activated K+ channel blocker, apamin, reversed the enhanced atrial arrhythmia inducibility. Apamin mitigated APD abbreviation without alteration of Ca2+ transient, implying an underlying mechanism of posttranslational modification of SK channels.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Animais , Apamina/farmacologia , Cálcio/metabolismo , Óxido Nítrico , Óxido Nítrico Sintase Tipo I , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa
14.
Int J Mol Sci ; 23(16)2022 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-36012704

RESUMO

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.


Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/farmacologia , Apamina , Ácido Elágico/farmacologia , Excipientes , Humanos , Lipídeos , Células MCF-7 , Tamanho da Partícula
15.
Biochem Biophys Res Commun ; 615: 157-162, 2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35643055

RESUMO

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.


Assuntos
Melatonina , Glândula Pineal , Canais de Potássio Cálcio-Ativados , Animais , Apamina/farmacologia , Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Melatonina/metabolismo , Glândula Pineal/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Pirazóis/farmacologia , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
16.
Drug Deliv ; 29(1): 1536-1548, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35612292

RESUMO

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-κß. Collectively, these findings showed that our optimized 2ME-INVA-APA could easily seep through the cell membrane and induce apoptosis in relatively low doses.


Assuntos
Apoptose , Neoplasias Pulmonares , 2-Metoxiestradiol/farmacologia , Células A549 , Apamina/farmacologia , Estradiol/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico
17.
Sci Rep ; 12(1): 5071, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35332235

RESUMO

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells have been proposed to regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 and PDGFRα and SK3 protein were reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Contractile experiments and ex vivo cystometry showed increased spontaneous contractions and transient contractions, respectively in CYP-treated bladders with a reduction of apamin sensitivity, that could be attributable to the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.


Assuntos
Cistite , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Animais , Apamina , Ciclofosfamida/efeitos adversos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
18.
J Sex Med ; 19(5): 697-710, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35321830

RESUMO

BACKGROUND: Activation of endothelial small conductance calcium-activated K+ channels (KCa2.3) and intermediate conductance calcium-activated K+ channels (KCa3.1) leads to vascular relaxation. We found endothelial KCa2.3 down-regulation in the corpus cavernosum diminishes erectile function. AIM: We hypothesized that in type-2 diabetic mice, the function of KCa2.3 and KCa1.1 channels is impaired in erectile tissue. METHODS: Erectile function was measured, and corpus cavernosum strips were mounted for functional studies and processed for qPCR and immunoblotting. OUTCOMES: Effects of type 2 diabetes on erectile function, expression and function of calcium-activated potassium channels. RESULTS: In anesthetized diabetic db/db mice, erectile function was markedly decreased compared to non-diabetic heterozygous db/+ mice, and the impairment was even more pronounced compared to normal C57BL/6 mice. qPCR revealed KCa2.3 and KCa1.1α channel expressions were upregulated in corpus cavernosum from db/db mice. Immunoblotting showed down-regulation of KCa2.3 channels in the corpus cavernosum from db/db mice. Acetylcholine relaxations were impaired while relaxations induced by the nitric oxide, donor SNP were unaltered in corpus cavernosum from db/db compared to C57BL/6 and db/+ mice. Apamin, a blocker of KCa2 channels, inhibited acetylcholine relaxation in corpus cavernosum from all experimental groups. In the presence of apamin, acetylcholine relaxation was markedly decreased in corpus cavernosum from db/db vs C57BL/6 and db/+ mice. An opener of KCa2 and KCa3.1 channels, NS309, potentiated acetylcholine relaxations in corpus cavernosum from db/+ and db/db mice. Iberiotoxin, a blocker of KCa1.1 channels, inhibited acetylcholine relaxation in corpus cavernosum from db/+ mice, while there was no effect in tissue from db/db mice. CLINICAL TRANSLATION: Erectile function in diabetic db/db mice was severely affected compared to heterozygous and control mice, findings suggesting the non-diabetic db/+ and diabetic db/db mice for translational purpose can be used for drug testing on, respectively, moderate and severe erectile dysfunction. The altered expressions and impaired acetylcholine relaxation in the presence of apamin compared to C57BL/6 mice may suggest decreased KCa1.1 channel function may underpin impaired endothelium-dependent relaxation and erectile dysfunction in diabetic db/db mice. STRENGTHS & LIMITATIONS: The present study provides a mouse model for type 2 diabetes to test moderate and severe erectile dysfunction drugs. Decreased KCa1.1 channel function contributes to erectile dysfunction, and it is a limitation that it is not supported by electrophysiological measurements. CONCLUSION: Our results suggest that the contribution of iberiotoxin-sensitive KCa1.1 channels to relaxation is reduced in the corpus cavernosum, while apamin-sensitive KCa2.3 channels appear upregulated. The impaired KCa1.1 channel function may contribute to the impaired erectile function in diabetic db/db mice. Comerma-Steffensen S, Prat-Duran J, Mogensen S, et al. Erectile Dysfunction and Altered Contribution of KCa1.1 and KCa2.3 Channels in the Penile Tissue of Type-2 Diabetic db/db Mice. J Sex Med 2022;19:697-710.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Disfunção Erétil , Acetilcolina/farmacologia , Animais , Apamina/metabolismo , Apamina/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pênis/irrigação sanguínea , Canais de Potássio Ativados por Cálcio de Condutância Baixa
19.
Psychopharmacology (Berl) ; 239(1): 253-266, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34982171

RESUMO

RATIONALE: The voltage-insensitive, small-conductance calcium-activated potassium (SK) channel is a key regulator of neuronal depolarization and is implicated in the pathophysiology of depressive disorders. OBJECTIVE: We ascertained whether the SK channel is impaired in the chronic unpredictable stress (CUS) model and whether it can serve as a molecular target of antidepressant action. METHODS: We assessed the depressive-like behavioral phenotype of CUS-exposed rats and performed post-mortem SK channel binding and activity-dependent zif268 mRNA analyses on their brains. To begin an assessment of SK channel subtypes involved, we examined the effects of genetic and pharmacological inhibition of the SK3 channel using conditional knockout mice and selective SK3 channel negative allosteric modulators (NAMs). RESULTS: We found that [125I]apamin binding to SK channels is increased in the prefrontal cortex and decreased in the hippocampus, an effect that was associated with reciprocal levels of zif268 mRNA transcripts indicating abnormal regional cell activity in this model. We found that genetic and pharmacological manipulations significantly decreased immobility in the forced swim test without altering general locomotor activity, a hallmark of antidepressant-like activity. CONCLUSIONS: Taken together, these findings link depression-related neural and behavioral pathophysiology with abnormal SK channel functioning and suggest that this can be reversed by the selective inhibition of SK3 channels.


Assuntos
Neurônios , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Animais , Antidepressivos/farmacologia , Apamina , Cálcio/metabolismo , Camundongos , Neurônios/metabolismo , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética
20.
J Sex Med ; 19(5): 697-710, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37057569

RESUMO

BACKGROUND: Activation of endothelial small conductance calcium-activated K+ channels (KCa2.3) and intermediate conductance calcium-activated K+ channels (KCa3.1) leads to vascular relaxation. We found endothelial KCa2.3 down-regulation in the corpus cavernosum diminishes erectile function. AIM: We hypothesized that in type-2 diabetic mice, the function of KCa2.3 and KCa1.1 channels is impaired in erectile tissue. METHODS: Erectile function was measured, and corpus cavernosum strips were mounted for functional studies and processed for qPCR and immunoblotting. OUTCOMES: Effects of type 2 diabetes on erectile function, expression and function of calcium-activated potassium channels. RESULTS: In anesthetized diabetic db/db mice, erectile function was markedly decreased compared to non-diabetic heterozygous db/+ mice, and the impairment was even more pronounced compared to normal C57BL/6 mice. qPCR revealed KCa2.3 and KCa1.1α channel expressions were upregulated in corpus cavernosum from db/db mice. Immunoblotting showed down-regulation of KCa2.3 channels in the corpus cavernosum from db/db mice. Acetylcholine relaxations were impaired while relaxations induced by the nitric oxide, donor SNP were unaltered in corpus cavernosum from db/db compared to C57BL/6 and db/+ mice. Apamin, a blocker of KCa2 channels, inhibited acetylcholine relaxation in corpus cavernosum from all experimental groups. In the presence of apamin, acetylcholine relaxation was markedly decreased in corpus cavernosum from db/db vs C57BL/6 and db/+ mice. An opener of KCa2 and KCa3.1 channels, NS309, potentiated acetylcholine relaxations in corpus cavernosum from db/+ and db/db mice. Iberiotoxin, a blocker of KCa1.1 channels, inhibited acetylcholine relaxation in corpus cavernosum from db/+ mice, while there was no effect in tissue from db/db mice. CLINICAL TRANSLATION: Erectile function in diabetic db/db mice was severely affected compared to heterozygous and control mice, findings suggesting the non-diabetic db/+ and diabetic db/db mice for translational purpose can be used for drug testing on, respectively, moderate and severe erectile dysfunction. The altered expressions and impaired acetylcholine relaxation in the presence of apamin compared to C57BL/6 mice may suggest decreased KCa1.1 channel function may underpin impaired endothelium-dependent relaxation and erectile dysfunction in diabetic db/db mice. STRENGTHS & LIMITATIONS: The present study provides a mouse model for type 2 diabetes to test moderate and severe erectile dysfunction drugs. Decreased KCa1.1 channel function contributes to erectile dysfunction, and it is a limitation that it is not supported by electrophysiological measurements. CONCLUSION: Our results suggest that the contribution of iberiotoxin-sensitive KCa1.1 channels to relaxation is reduced in the corpus cavernosum, while apamin-sensitive KCa2.3 channels appear upregulated. The impaired KCa1.1 channel function may contribute to the impaired erectile function in diabetic db/db mice.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Disfunção Erétil , Canais de Potássio Cálcio-Ativados , Masculino , Humanos , Camundongos , Animais , Acetilcolina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Experimental/complicações , Apamina/farmacologia , Apamina/metabolismo , Camundongos Endogâmicos C57BL , Pênis/irrigação sanguínea , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Potássio Cálcio-Ativados/farmacologia
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