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1.
Sci Rep ; 11(1): 16268, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381120

RESUMO

Volume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+, and they restrain the excitability of smooth muscle at low levels and prevents the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα+ cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα+ cells is causative in the development of DO after SCI.


Assuntos
Contração Muscular/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Traumatismos da Medula Espinal/complicações , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologia , Animais , Apamina/metabolismo , Apoptose , Expressão Gênica , Camundongos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Traumatismos da Medula Espinal/genética , Bexiga Urinária/citologia , Bexiga Urinária/patologia , Bexiga Urinária Hiperativa/fisiopatologia
2.
PLoS One ; 16(7): e0255161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34293062

RESUMO

Bee venom is a natural substance produced by worker bees. The aim of this research paper is to determine the characteristics of Anatolian bee venom by evaluating its chemical content and microbiological properties. Physical, chemical and microbiological analyses were performed on 25 bee venom samples from different areas of Anatolia, Turkey. Data obtained by 3-replicate studies were evaluated with normality and one-way and two-way ANOVA / Tukey tests. Chemical analyses of the bee venoms revealed average melittin, apamin, and phospholipase A2 contents of 40.57%, 2.12% and 13.67%, respectively. The results suggest that Anatolian bee venom has a high phospholipase A2 content compared to the previous literature. The results for apamin content were similar to those reported in other countries. Melittin content was within the range of standard values. Bee venom samples were also observed to have a high sugar content, associated with pollen and nectar contamination. Total aerobic mesophilic bacteria counts revealed no microbial development in 11 samples of bee venom. Staphylococcus aureus was not detected in any sample. A low microbial load was associated with a high phospholipase A2 content in the bee venom composition, thus contributing to its antimicrobial character. This study presents an examination of Anatolian bee venom in terms of chemical content and microbial quality. The examination of other components in addition to phospholipase A2, melittin and apamin in future studies, together with an analysis of antimicrobial properties will further our understanding of Anatolian bee venom.


Assuntos
Venenos de Abelha/química , Abelhas/microbiologia , Animais , Apamina/análise , Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Frutose/análise , Glucose/análise , Umidade , Meliteno/análise , Fosfolipases A2/análise , Manejo de Espécimes , Sacarose/análise , Turquia
3.
Toxins (Basel) ; 13(5)2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067049

RESUMO

Prostate cancer is one of the most common cancers in men. Despite the development of a variety of therapeutic agents to treat either metastatic hormone-sensitive prostate cancer, advanced prostate cancer, or nonmetastatic/metastatic castration-resistant prostate cancer, the progression or spread of the disease often cannot be avoided. Additionally, the development of resistance of prostate cancer cells to available therapeutic agents is a well-known problem. Despite extensive and cost-intensive research over decades, curative therapy for metastatic prostate cancer is still not available. Therefore, additional therapeutic agents are still needed. The animal kingdom offers a valuable source of natural substances used for the treatment of a variety of diseases. Bee venom of the honeybee is a mixture of many components. It contains proteins acting as enzymes such as phospholipase A2, smaller proteins and peptides such as melittin and apamin, phospholipids, and physiologically active amines such as histamine, dopamine, and noradrenaline. Melittin has been shown to induce apoptosis in different cancer cell lines, including prostate cancer cell lines. It also influences cell proliferation, angiogenesis, and necrosis as well as motility, migration, metastasis, and invasion of tumour cells. Hence, it represents an interesting anticancer agent. In this review article, studies about the effect of bee venom components on prostate cancer cells are discussed. An electronic literature research was performed utilising PubMed in February 2021. All scientific publications, which examine this interesting subject, are discussed. Furthermore, the different types of application of these promising substances are outlined. The studies clearly indicate that bee venom or melittin exhibited anticancer effects in various prostate cancer cell lines and in xenografts. In most of the studies, a combination of bee venom or the modified melittin with another molecule was utilised in order to avoid side effects and, additionally, to target selectively the prostate cancer cells or the surrounding tissue. The studies showed that systemic side effects and unwanted damage to healthy tissue and organs could be minimised when the anticancer drug was not activated until binding to the cancer cells or the surrounding tissue. Different targets were used, such as the matrix metalloproteinase 2, hormone receptors expressed by prostate cancer cells, the extracellular domain of PSMA, and the fibroblast activation protein occurring in the stroma of prostate cancer cells. Another approach used loaded phosphate micelles, which were cleaved by the enzyme secretory phospholipase A2 produced by prostate cancer cells. In a totally different approach, targeted nanoparticles containing the melittin gene were used for prostate cancer gene therapy. By the targeted nonviral gene delivery, the gene encoding melittin was delivered to the prostate cancer cells without systemic side effects. This review of the scientific literature reveals totally different approaches using bee venom, melittin, modified melittin, or protoxin as anticancer agents. The toxic agents acted through several different mechanisms to produce their anti-prostate cancer effects. These mechanisms are not fully understood yet and more experimental studies are necessary to reveal the complete mode of action. Nevertheless, the researchers have conducted pioneering work. Based on these results, further experimental and clinical studies about melittin and modifications of this interesting agent deriving from nature are necessary and could possibly lead to a complementary treatment option for prostate cancer.


Assuntos
Antineoplásicos/farmacologia , Venenos de Abelha/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apamina/isolamento & purificação , Apamina/farmacologia , Apoptose/efeitos dos fármacos , Venenos de Abelha/administração & dosagem , Venenos de Abelha/química , Abelhas , Humanos , Masculino , Meliteno/isolamento & purificação , Meliteno/farmacologia , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/farmacologia , Neoplasias da Próstata/patologia
5.
J Physiol ; 599(11): 2933-2951, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33651437

RESUMO

KEY POINTS: NMDA receptors (NMDARs) expressed by dopamine neurons of the ventral tegmental area (VTA) play a central role in glutamate synapse plasticity, neuronal firing and adaptative behaviours. The NMDAR surface dynamics shapes synaptic adaptation in hippocampal networks, as well as associative memory. We investigated the basic properties and role of the NMDAR surface dynamics on cultured mesencephalic and VTA dopamine neurons in rodents. Using a combination of single molecule imaging and electrophysiological recordings, we demonstrate that NMDARs are highly diffusive at the surface of mesencephalic dopamine neurons. Unexpectedly, the NMDAR membrane dynamics per se regulates the firing pattern of VTA dopaminergic neurons, probably through a functional interplay between NMDARs receptors and small-conductance calcium-dependent potassium (SK) channels. ABSTRACT: Midbrain dopaminergic (DA) neurons play a central role in major physiological brain functions, and their dysfunctions have been associated with neuropsychiatric diseases. The activity of midbrain DA neurons is controlled by ion channels and neurotransmitter receptors, such as the glutamate NMDA receptor (NMDAR) and small-conductance calcium-dependent potassium (SK) channels. However, the cellular mechanisms through which these channels tune the firing pattern of midbrain DA neurons remain unclear. Here, we investigated whether the surface dynamics and distribution of NMDARs tunes the firing pattern of midbrain DA neurons. Using a combination of single molecule imaging and electrophysiological recordings, we report that NMDARs are highly diffusive at the surface of cultured midbrain DA neurons from rodents and humans. Reducing acutely the NMDAR membrane dynamics, which leaves the ionotropic function of the receptor intact, robustly altered the firing pattern of midbrain DA neurons without altering synaptic glutamatergic transmission. The reduction of NMDAR surface dynamics reduced apamin (SK channel blocker)-induced firing change and the distribution of SK3 channels in DA neurons. Together, these data show that the surface dynamics of NMDAR, and not solely its ionotropic function, tune the firing pattern of midbrain DA neurons partly through a functional interplay with SK channel function.


Assuntos
Neurônios Dopaminérgicos , Receptores de N-Metil-D-Aspartato , Potenciais de Ação , Apamina , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo , Receptores de N-Metil-D-Aspartato/metabolismo , Área Tegmentar Ventral
6.
Am J Physiol Heart Circ Physiol ; 320(4): H1456-H1469, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33635168

RESUMO

Ventricular arrhythmia (VA) is the major cause of death in patients with left ventricular (LV) hypertrophy and/or acute ischemia. We hypothesized that apamin, a blocker of small-conductance Ca2+-activated K+ (SK) channels, alters Ca2+ handling and exhibits anti-arrhythmic effects in ventricular myocardium. Spontaneous hypertensive rats were used as a model of LV hypertrophy. A dual optical mapping of membrane potential (Vm) and intracellular calcium (Cai) was performed during global hypoxia (GH) on the Langendorff perfusion system. The majority of pacing-induced VAs during GH were initiated by triggered activities. Pretreatment of apamin (100 nmol/L) significantly inhibited the VA inducibility. Compared with SK channel blockers (apamin and NS8593), non-SK channel blockers (glibenclamide and 4-AP) did not exhibit anti-arrhythmic effects. Apamin prevented not only action potential duration (APD80) shortening (-18.7 [95% confidence interval, -35.2 to -6.05] ms vs. -2.75 [95% CI, -10.45 to 12.65] ms, P = 0.04) but also calcium transient duration (CaTD80) prolongation (14.52 [95% CI, 8.8-20.35] ms vs. 3.85 [95% CI, -3.3 to 12.1] ms, P < 0.01), thereby reducing CaTD80 - APD80, which denotes "Cai/Vm uncoupling" (33.22 [95% CI, 22-48.4] ms vs. 6.6 [95% CI, 0-14.85] ms, P < 0.01). The reduction of Cai/Vm uncoupling was attributable to less prolonged Ca2+ decay constant and suppression of diastolic Cai increase by apamin. The inhibition of VA inducibility and changes in APs/CaTs parameters caused by apamin was negated by the addition of ouabain, an inhibitor of Na+/K+ pump. Apamin attenuates APD shortening, Ca2+ handling abnormalities, and Cai/Vm uncoupling, leading to inhibition of VA occurrence in hypoxic hypertrophied hearts.NEW & NOTEWORTHY We demonstrated that hypoxia-induced ventricular arrhythmias were mainly initiated by Ca2+-loaded triggered activities in hypertrophied hearts. The blockades of small-conductance Ca2+-activated K+ channels, especially "apamin," showed anti-arrhythmic effects by alleviation of not only action potential duration shortening but also Ca2+ handling abnormalities, most notably the "Ca2+/voltage uncoupling."


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Sinalização do Cálcio/efeitos dos fármacos , Cardiomegalia/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Hipóxia/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Apamina/farmacologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Estimulação Cardíaca Artificial , Cardiomegalia/complicações , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Hipóxia/complicações , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Preparação de Coração Isolado , Masculino , Ratos Endogâmicos SHR , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Fatores de Tempo
7.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525680

RESUMO

Kynurenic acid (KYNA, 4-oxoquinoline-2-carboxylic acid), an intermediate of the tryptophan metabolism, has been recognized to exert different neuroactive actions; however, the need of how it or its aminoalkylated amide derivative N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-oxo-1,4-dihydroquinoline-2-carboxamide (KYNA-A4) exerts any effects on ion currents in excitable cells remains largely unmet. In this study, the investigations of how KYNA and other structurally similar KYNA derivatives have any adjustments on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were performed by patch-clamp technique. KYNA or KYNA-A4 increased the amplitude of M-type K+ current (IK(M)) and concomitantly enhanced the activation time course of the current. The EC50 value required for KYNA- or KYNA-A4 -stimulated IK(M) was yielded to be 18.1 or 6.4 µM, respectively. The presence of KYNA or KYNA-A4 shifted the relationship of normalized IK(M)-conductance versus membrane potential to more depolarized potential with no change in the gating charge of the current. The voltage-dependent hysteretic area of IK(M) elicited by long-lasting triangular ramp pulse was observed in GH3 cells and that was increased during exposure to KYNA or KYNA-A4. In cell-attached current recordings, addition of KYNA raised the open probability of M-type K+ channels, along with increased mean open time of the channel. Cell exposure to KYNA or KYNA-A4 mildly inhibited delayed-rectifying K+ current; however, neither erg-mediated K+ current, hyperpolarization-activated cation current, nor voltage-gated Na+ current in GH3 cells was changed by KYNA or KYNA-A4. Under whole-cell, current-clamp recordings, exposure to KYNA or KYNA-A4 diminished the frequency of spontaneous action potentials; moreover, their reduction in firing frequency was attenuated by linopirdine, yet not by iberiotoxin or apamin. In hippocampal mHippoE-14 neurons, the addition of KYNA also increased the IK(M) amplitude effectively. Taken together, the actions presented herein would be one of the noticeable mechanisms through which they modulate functional activities of excitable cells occurring in vivo.


Assuntos
Hipocampo/fisiologia , Canais de Potássio KCNQ/efeitos dos fármacos , Ácido Cinurênico/farmacologia , Animais , Apamina/farmacologia , Linhagem Celular , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Indóis/farmacologia , Ácido Cinurênico/química , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Piridinas/farmacologia , Ratos
8.
J Physiol ; 599(2): 507-520, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-31667845

RESUMO

KEY POINTS: Glutamatergic NMDA receptors (NMDARs) and small conductance Ca2+ -activated K+ (SK) channels are critical synaptic and intrinsic mechanisms, respectively, that regulate the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). In this work, we investigated whether NMDARs and SK channels in MNNs are functionally coupled, and whether an altered coupling may contribute to exacerbated neuronal activity in this condition. We report that NMDARs and SK channels form a functional Ca2+ -dependent negative feedback loop that restrains the excitatory effect on membrane potential and firing activity evoked by NMDAR activation. The negative feedback loop between NMDARs and SK channels was blunted or absent in MNNs of heart failure (HF) rats. These results help us better understand how synaptic and intrinsic mechanisms regulate hypothalamic neuronal activity, as well as how changes in the interaction among these disparate mechanisms contribute to altered neuronal activity during prevalent neurogenic cardiovascular diseases. ABSTRACT: Glutamatergic NMDA receptors (NMDARs) and small conductance Ca2+ -activated K+ (SK) channels are critical synaptic and intrinsic mechanisms, respectively, that regulate the activity of hypothalamic magnocellular neurosecretory neurons (MNNs), both under physiological and pathological states, such as lactation and heart failure (HF). However, whether NMDARs and SK channels in MNNs are functionally coupled, and whether changes in this coupling contribute to exacerbated neuronal activity during HF is at present unknown. In the present study, we addressed these questions using patch-clamp electrophysiology and confocal Ca2+ imaging in a rat model of ischaemic HF. We found that in MNNs of sham rats, blockade of SK channels with apamin (200 nM) significantly increased the magnitude of an NMDAR-evoked current (INMDA ). We also observed that blockade of SK channels potentiated NMDAR-evoked firing, and abolished spike frequency adaptation in MNNs from sham, but not HF rats. Importantly, a larger INMDA -ΔCa2+ response was observed under basal conditions in HF compared to sham rats. Finally, we found that dialysing recorded cells with the Ca2+ chelator BAPTA (10 mM) increased the magnitude of INMDA in MNNs from both sham and HF rats, and occluded the effects of apamin in the former. Together our studies demonstrate that in MNNs, NMDARs and SK channels are functionally coupled, forming a local negative feedback loop that restrains the excitatory effect evoked by NMDAR activation. Moreover, our studies also support a blunted NMDAR-SK channel coupling in MNNs of HF rats, establishing it as a pathophysiological mechanism contributing to exacerbated hypothalamic neuronal activity during this prevalent neurogenic cardiovascular disease.


Assuntos
Insuficiência Cardíaca , Receptores de N-Metil-D-Aspartato , Animais , Apamina , Feminino , Hipotálamo/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa
9.
Molecules ; 25(23)2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33287398

RESUMO

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.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Antioxidantes/fisiologia , Apamina/farmacologia , Apoptose/efeitos dos fármacos , Inflamação/tratamento farmacológico , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Sepse/tratamento farmacológico , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Mol Biol Rep ; 47(11): 9013-9019, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33174081

RESUMO

Multiple sclerosis (MS) is a chronic debilitating disease that attacks the central nervous system. This study aims to investigate miR-219 and miR-155-3p expression levels involved in the myelination process following the administration of apamin peptide in the model of multiple sclerosis disease. Forty-four 8 week C57BL/6 male mice (22 ± 5 g) randomly divided into six groups. Apamin (100 µg/kg/BW) was administered intraperitoneally as a co-treatment during phase I (demyelination) or post-treatment phase II (remyelination) twice a week in cuprizone induced MS model. At the end of study myelin content and microRNA expression levels were measured with LFB staining and quantitative Real-Time PCR method, respectively. It was observed that the intended microRNAs were dysregulated during the different phases of disease induction. After 6 weeks of cuprizone exposure, miR-219 downregulated in phase I in comparison with the negative control. On the other hand, the apamin co-treatment significantly inhibit the miR-155-3p upregulation during the phase I as compared with the cuprizone group (p < 0.0001). Apamin has more impact on the miR155-3p reduction in phase I than miR-219 elevation in phase II. It could be considered as a therapeutic option for decreasing plaque formation during the exacerbation phase of the MS disease. Apamin has more impact on the miR155-3p reduction in phase I than miR-219 elevation in phase II. It could be considered as a therapeutic option for decreasing plaque formation during the exacerbation phase of the MS disease.


Assuntos
Apamina/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Esclerose Múltipla/genética , Animais , Cuprizona , Doenças Desmielinizantes/genética , Masculino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/induzido quimicamente , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/genética
11.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751347

RESUMO

The transient receptor potential-melastatin 8 (TRPM8) is a non-selective Ca2+-permeable channel, activated by cold, membrane depolarization, and different cooling compounds. TRPM8 expression has been found in gut mucosal, submucosal, and muscular nerve endings. Although TRPM8 plays a role in pathological conditions, being involved in visceral pain and inflammation, the physiological functions in the digestive system remain unclear as yet. The aims of the present study were: (i) to verify the TRPM8 expression in human distal colon; (ii) to examine the effects of TRPM8 activation on colonic contractility; (iii) to characterize the mechanism of action. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were used to analyze TRPM8 expression. The responses of human colon circular strips to different TRPM8 agonists [1-[Dialkyl-phosphinoyl]-alkane (DAPA) 2-5, 1-[Diisopropyl-phosphinoyl]-alkane (DIPA) 1-7, DIPA 1-8, DIPA 1-9, DIPA 1-10, and DIPA 1-12) were recorded using a vertical organ bath. The biomolecular analysis revealed gene and protein expression of TRPM8 in both mucosal and smooth muscle layers. All the agonists tested, except-DIPA 1-12, produced a concentration-dependent decrease in spontaneous contraction amplitude. The effect was significantly antagonized by 5-benzyloxytryptamine, a TRPM8 antagonist. The DIPA 1-8 agonist resulted in the most efficacious and potent activation among the tested molecules. The DIPA 1-8 effects were not affected by tetrodotoxin, a neural blocker, but they were significantly reduced by tetraethylammonium chloride, a non-selective blocker of K+ channels. Moreover, iberiotoxin, a blocker of the large-conductance Ca2+-dependent K+-channels, but not apamin, a blocker of small-conductance Ca2+-dependent K+ channels, significantly reduced the inhibitory DIPA 1-8 actions. The results of the present study demonstrated that TRPM8 receptors are also expressed in human distal colon in healthy conditions and that ligand-dependent TRPM8 activation is able to reduce the colonic spontaneous motility, probably by the opening of the large-conductance Ca2+-dependent K+-channels.


Assuntos
Colo/metabolismo , Mucosa Intestinal/metabolismo , Contração Muscular/genética , Músculo Liso/metabolismo , Canais de Cátion TRPM/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apamina/farmacologia , Colo/efeitos dos fármacos , Feminino , Expressão Gênica , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Peptídeos/farmacologia , Ácidos Fosfínicos/farmacologia , Serotonina/análogos & derivados , Serotonina/farmacologia , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia , Técnicas de Cultura de Tecidos
12.
Biophys J ; 119(3): 690-704, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32668235

RESUMO

Cardiac small conductance Ca2+-activated K+ (SK) channels are activated solely by Ca2+, but the SK current (ISK) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under ß-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca2+/Mg2+ block. Thus, Ca2+ has a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the effect of ISK rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of ISK during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca2+] reached ∼10 µM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] decreased further. We used a six-state gating model combined with intrinsic and Ca2+/Mg2+-dependent rectification to simulate ISK and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of ISK recording during AP clamp showing that intrinsic rectification limits ISK at high Vm during the early and plateau phase of APs. Furthermore, the initial rise of Ca2+ transients activates, but higher [Ca2+] blocks SK channels, yielding a transient outward-like ISK trajectory. During the decay phase of Ca2+, the Ca2+-dependent block is released, causing ISK to rise again and contribute to repolarization. Therefore, ISK is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.


Assuntos
Miócitos Cardíacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Potenciais de Ação , Animais , Apamina , Ventrículos do Coração , Ratos
13.
Molecules ; 25(14)2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32664327

RESUMO

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IKCa) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IKCa channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K+ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K+ channel blockers: iberiotoxin (BKca channel blocker), apamin (SKca channel blocker), and charybdotoxin (IKca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC20).


Assuntos
Basidiomycota/química , Artérias Mesentéricas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vasodilatação/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Apamina/farmacologia , Charibdotoxina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Peptídeos/farmacologia , Phellinus , Fosforilação/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Tetraetilamônio/farmacologia , Vasoconstrição/efeitos dos fármacos
14.
Int J Mol Sci ; 21(12)2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32560481

RESUMO

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.


Assuntos
Apamina/farmacologia , Lipopolissacarídeos/efeitos adversos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Citocinas/metabolismo , Suscetibilidade a Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , NF-kappa B/metabolismo , Ratos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo
15.
J Ethnopharmacol ; 257: 112860, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32289477

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF STUDY: The 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 METHODS: To 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. RESULTS: BV 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. CONCLUSIONS: These results suggest that the APM from BV might be useful for the treatment of gouty arthritis due to its anti-inflammatory activities.


Assuntos
Anti-Inflamatórios/farmacologia , Apamina/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Gotosa/prevenção & controle , Venenos de Abelha/farmacologia , Articulações/efeitos dos fármacos , Animais , Apamina/isolamento & purificação , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Artrite Gotosa/patologia , Venenos de Abelha/química , Citocinas/genética , Citocinas/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Mediadores da Inflamação/metabolismo , Articulações/metabolismo , Articulações/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Transdução de Sinais , Ácido Úrico
16.
Toxins (Basel) ; 12(3)2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204567

RESUMO

Bee venom is a natural toxin produced by honeybees and plays an important role in defending bee colonies. Bee venom has several kinds of peptides, including melittin, apamin, adolapamine, and mast cell degranulation peptides. Apamin accounts for about 2%-3% dry weight of bee venom and is a peptide neurotoxin that contains 18 amino acid residues that are tightly crosslinked by two disulfide bonds. It is well known for its pharmacological functions, which irreversibly block Ca2+-activated K+ (SK) channels. Apamin regulates gene expression in various signal transduction pathways involved in cell development. The aim of this study was to review the current understanding of apamin in the treatment of apoptosis, fibrosis, and central nervous system diseases, which are the pathological processes of various diseases. Apamin's potential therapeutic and pharmacological applications are also discussed.


Assuntos
Apamina/uso terapêutico , Aterosclerose/tratamento farmacológico , Venenos de Abelha/química , Sistema Nervoso Central/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Apamina/isolamento & purificação , Apoptose/efeitos dos fármacos , Aterosclerose/metabolismo , Sistema Nervoso Central/patologia , Citocinas/antagonistas & inibidores , Fibrose , Humanos , Cirrose Hepática/patologia
17.
J Physiol ; 598(14): 2847-2873, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-30771223

RESUMO

KEY POINTS: Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+ ] in a biphasic manner. Submicromolar [Ca2+ ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+ /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+ ]i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. ABSTRACT: Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 µmol L-1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L-1 ), which was eliminated by inhibition of PKA (1 µmol L-1 ). Beta-adrenergic stimulation (isoproterenol, 100 nmol L-1 ) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+ /voltage-dependent inhibition of the channels at high [Ca2+ ] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.


Assuntos
Miócitos Cardíacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Animais , Apamina , Cardiomegalia/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
18.
Heart Rhythm ; 17(2): 332-340, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31513946

RESUMO

BACKGROUND: Ondansetron, a widely prescribed antiemetic, has been implicated in drug-induced long QT syndrome. Recent patch clamp experiments have shown that ondansetron inhibits the apamin-sensitive small conductance calcium-activated potassium current (IKAS). OBJECTIVE: The purpose of this study was to determine whether ondansetron causes action potential duration (APD) prolongation by IKAS inhibition. METHODS: Optical mapping was performed in rabbit hearts with pacing-induced heart failure (HF) and in normal hearts before and after ondansetron (100 nM) infusion. APD at 80% repolarization (APD80) and arrhythmia inducibility were determined. Additional studies with ondansetron were performed in normal hearts perfused with hypokalemic Tyrode's (2.4 mM) solution before or after apamin administration. RESULTS: The corrected QT interval in HF was 326 ms (95% confidence interval [CI] 306-347 ms) at baseline and 364 ms (95% CI 351-378 ms) after ondansetron infusion (P < .001). Ondansetron significantly prolonged APD80 in the HF group and promoted early afterdepolarizations, steepened the APD restitution curve, and increased ventricular vulnerability. Ventricular fibrillation was not inducible in HF ventricles at baseline, but after ondansetron infusion, ventricular fibrillation was induced in 5 of the 7 ventricles (P = .021). In hypokalemia, apamin prolonged APD80 from 163 ms (95% CI 146-180 ms) to 180 ms (95% CI 156-204 ms) (P = .018). Subsequent administration of ondansetron failed to further prolong APD80 (180 ms [95% CI 156-204 ms] vs 179 ms [95% CI 165-194 ms]; P = .789). The results were similar when ondansetron was administered first, followed by apamin. CONCLUSION: Ondansetron is a specific IKAS blocker at therapeutic concentrations. Ondansetron may prolong the QT interval in HF by inhibiting small conductance calcium-activated potassium channels, which increases the vulnerability to ventricular arrhythmias.


Assuntos
Estimulação Cardíaca Artificial , Insuficiência Cardíaca/terapia , Ventrículos do Coração/fisiopatologia , Ondansetron/farmacologia , Fibrilação Ventricular/complicações , Potenciais de Ação , Animais , Apamina/farmacologia , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Técnicas de Patch-Clamp , Coelhos , Antagonistas da Serotonina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Fibrilação Ventricular/fisiopatologia
19.
Cereb Cortex ; 30(2): 563-574, 2020 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-31188425

RESUMO

Disruption of attention is an early and disabling symptom of Alzheimer's disease (AD). The underlying cellular mechanisms are poorly understood and treatment options for patients are limited. These early attention deficits are evident in the TgCRND8 mouse, a well-established murine model of AD that recapitulates several features of the disease. Here, we report severe impairment of the nicotinic receptor-mediated excitation of prefrontal attentional circuitry in TgCRND8 mice relative to wild-type littermate controls. We demonstrate that this impairment can be remedied by apamin, a bee venom neurotoxin peptide that acts as a selective antagonist to the SK family of calcium-sensitive potassium channels. We probe this seeming upregulation of calcium-sensitive inhibition and find that the attenuated nicotinic firing rates in TgCRND8 attention circuits are mediated neither by greater cellular calcium signals nor by elevated SK channel expression. Instead, we find that TgCRND8 mice show enhanced functional coupling of nicotinic calcium signals to inhibition. This SK-mediated inhibition exerts a powerful negative feedback on nicotinic excitation, dampening attention-relevant signaling in the TgCRND8 brain. These mechanistic findings identify a new cellular target involved in the modulation of attention and a novel therapeutic target for early attention deficits in AD.


Assuntos
Doença de Alzheimer/fisiopatologia , Apamina/administração & dosagem , Córtex Pré-Frontal/fisiologia , Células Piramidais/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores
20.
Toxicol Appl Pharmacol ; 384: 114799, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678606

RESUMO

The present study investigated the vasorelaxant effects of sitagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor in aortic rings pre-contracted with phenylephrine (Phe). Sitagliptin induced vasorelaxation in a concentration-dependent manner but the inhibition of voltage-dependent K+ (Kv) channels by pretreatment with 4-aminopyridine (4-AP) effectively reduced this effect. By contrast, the inhibition of inward rectifier K+ (Kir) channels by pretreatment with barium (Ba2+), large-conductance calcium (Ca2+)-activated K+ (BKCa) channels with paxilline, and adenosine triphosphate (ATP)-sensitive K+ (KATP) channels with glibenclamide did not change this effect. Although the application of SQ 22536, which is an adenylyl cyclase inhibitor, also did not change this effect, treatment with KT 5720, a protein kinase A (PKA) inhibitor, effectively reduced the vasorelaxant effects of sitagliptin. ODQ, which is a guanylyl cyclase inhibitor, and KT 5823, a protein kinase G (PKG) inhibitor, did not impact the effect. Furthermore, neither the inhibition of Ca2+ channels by pretreatment with nifedipine nor the inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps by pretreatment with thapsigargin changed the effect. Similarly, the effects of sitagliptin were not altered by eliminating the endothelium, by pretreatment with a nitric oxide (NO) synthase inhibitor (L-NAME), or by inhibition of small- and intermediate-conductance Ca2+-activated K+ channels (SKCa and IKCa) using apamin and TRAM-34. Taken together, these results suggest that sitagliptin induces vasorelaxation by inhibiting both membrane potential (Em)-dependent and -independent vasoconstriction and activating PKA and Kv channels independently of PKG signaling pathways, other K+ channels, SERCA pumps, and the endothelium.


Assuntos
Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fosfato de Sitagliptina/efeitos adversos , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica , Apamina/farmacologia , Carbazóis/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endotélio Vascular/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Fenilefrina/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Pirazóis/farmacologia , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Vasoconstrição/efeitos dos fármacos
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