The Endocannabinoid Peptide RVD-Hemopressin Is a TRPV1 Channel Blocker.

Neurotransmission is critical for brain function, allowing neurons to communicate through neurotransmitters and neuropeptides. RVD-hemopressin (RVD-Hp), a novel peptide identified in noradrenergic neurons, modulates cannabinoid receptors CB1 and CB2. Unlike hemopressin (Hp), which induces anxiogenic behaviors via transient receptor potential vanilloid 1 (TRPV1) activation, RVD-Hp counteracts these effects, suggesting that it may block TRPV1. This study investigates RVD-Hp's role as a TRPV1 channel blocker using HEK293 cells expressing TRPV1-GFP. Calcium imaging and patch-clamp recordings demonstrated that RVD-Hp reduces TRPV1-mediated calcium influx and TRPV1 ion currents. Molecular docking and dynamics simulations indicated that RVD-Hp interacts with TRPV1's selectivity filter, forming stable hydrogen bonds and van der Waals contacts, thus preventing ion permeation. These findings highlight RVD-Hp's potential as a therapeutic agent for conditions involving TRPV1 activation, such as pain and anxiety.

Knockin' on Egg's Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species.

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype-phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

TRPM8 Channel Promotes the Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells.

Various families of ion channels have been characterized in mesenchymal stem cells (MSCs), including some members of transient receptor potential (TRP) channels family. TRP channels are involved in critical cellular processes as differentiation and cell proliferation. Here, we analyzed the expression of TRPM8 channel in human bone marrow MSCs (hBM-MSCs), and its relation with osteogenic differentiation. Patch-clamp recordings showed that hBM-MSCs expressed outwardly rectifying currents which were increased by exposure to 500 µM menthol and were partially inhibited by 10 µM of BCTC, a TRPM8 channels antagonist. Additionally, we have found the expression of TRPM8 by RT-PCR and western blot. We also explored the TRPM8 localization in hBM-MSCs by immunofluorescence using confocal microscopy. Remarkably, hBM-MSCs treatment with 100 µM of menthol or 10 µM of icilin, TRPM8 agonists, increases osteogenic differentiation. Conversely, 20 µM of BCTC, induced a decrease of osteogenic differentiation. These results suggest that TRPM8 channels are functionally active in hBM-MSCs and have a role in cell differentiation.

Hypoxic Regulation of the Large-Conductance, Calcium and Voltage-Activated Potassium Channel, BK.

Hypoxia is a condition characterized by a reduction of cellular oxygen levels derived from alterations in oxygen balance. Hypoxic events trigger changes in cell-signaling cascades, oxidative stress, activation of pro-inflammatory molecules, and growth factors, influencing the activity of various ion channel families and leading to diverse cardiovascular diseases such as myocardial infarction, ischemic stroke, and hypertension. The large-conductance, calcium and voltage-activated potassium channel (BK) has a central role in the mechanism of oxygen (O2) sensing and its activity has been related to the hypoxic response. BK channels are ubiquitously expressed, and they are composed by the pore-forming α subunit and the regulatory subunits ß (ß1-ß4), γ (γ1-γ4), and LINGO1. The modification of biophysical properties of BK channels by ß subunits underly a myriad of physiological function of these proteins. Hypoxia induces tissue-specific modifications of BK channel α and ß subunits expression. Moreover, hypoxia modifies channel activation kinetics and voltage and/or calcium dependence. The reported effects on the BK channel properties are associated with events such as the increase of reactive oxygen species (ROS) production, increases of intracellular Calcium ([Ca2+]i), the regulation by Hypoxia-inducible factor 1α (HIF-1α), and the interaction with hemeproteins. Bronchial asthma, chronic obstructive pulmonary diseases (COPD), and obstructive sleep apnea (OSA), among others, can provoke hypoxia. Untreated OSA patients showed a decrease in BK-ß1 subunit mRNA levels and high arterial tension. Treatment with continuous positive airway pressure (CPAP) upregulated ß1 subunit mRNA level, decreased arterial pressures, and improved endothelial function coupled with a reduction in morbidity and mortality associated with OSA. These reports suggest that the BK channel has a role in the response involved in hypoxia-associated hypertension derived from OSA. Thus, this review aims to describe the mechanisms involved in the BK channel activation after a hypoxic stimulus and their relationship with disorders like OSA. A deep understanding of the molecular mechanism involved in hypoxic response may help in the therapeutic approaches to treat the pathological processes associated with diseases involving cellular hypoxia.

Ion Channel Function During Oocyte Maturation and Fertilization.

The proper maturation of both male and female gametes is essential for supporting fertilization and the early embryonic divisions. In the ovary, immature fully-grown oocytes that are arrested in prophase I of meiosis I are not able to support fertilization. Acquiring fertilization competence requires resumption of meiosis which encompasses the remodeling of multiple signaling pathways and the reorganization of cellular organelles. Collectively, this differentiation endows the egg with the ability to activate at fertilization and to promote the egg-to-embryo transition. Oocyte maturation is associated with changes in the electrical properties of the plasma membrane and alterations in the function and distribution of ion channels. Therefore, variations on the pattern of expression, distribution, and function of ion channels and transporters during oocyte maturation are fundamental to reproductive success. Ion channels and transporters are important in regulating membrane potential, but also in the case of calcium (Ca2+), they play a critical role in modulating intracellular signaling pathways. In the context of fertilization, Ca2+ has been shown to be the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. These early events include the block of polyspermy, the completion of meiosis and the transition to the embryonic mitotic divisions. In this review, we discuss the role of ion channels during oocyte maturation, fertilization and early embryonic development. We will describe how ion channel studies in Xenopus oocytes, an extensively studied model of oocyte maturation, translate into a greater understanding of the role of ion channels in mammalian oocyte physiology.

Conotoxins as Tools to Understand the Physiological Function of Voltage-Gated Calcium (CaV) Channels.

Voltage-gated calcium (CaV) channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1-1.4; CaV2.1-2.3) and Low-voltage activated (LVA, CaV3.1-3.3). HVA channels are highly expressed in brain (neurons), heart, and adrenal medulla (chromaffin cells), among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, "conopeptides", which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

[Factors associated with access to health services prior to pregnancy by pregnant adolescents]. / Fatores associados ao acesso anterior à gestação a serviços de saúde por adolescentes gestantes.

OBJECTIVE: To assess determinants of access to primary care services prior to pregnancy by pregnant adolescents. METHODS: Theory-based cross-sectional study conducted to examine access to heath services at five dimensions: geographic, economic, administrative, psychosocial and information. There were included in the study 200 first-time pregnant adolescents (aged 10 to 19 years) who attended a primary care unit in the municipality of Indaiatuba, Southeastern Brazil, in 2003. Data was collected using a semi-structured questionnaire applied in face-to-face interviews conducted just before their first pre-natal care visit. Data analyses were performed through Person's chi-square or Fisher's exact test, and by a multivariate logistic regression model including all five dimensions of access. RESULTS: More than half (63.7%) of the adolescents utilized some gynecological care service. Information (43.8%) or psychosocial feelings of embarrassment or fear (37.0%) barriers were the most frequent reasons given for not having sought care before. The main barrier to health service access was related to the psychosocial dimension, reported by 77.0% of the adolescents studied. CONCLUSIONS: Among all barriers to health service access, the most important were those related to the psychosocial dimension. There is a need for new strategies to facilitate access to health services by adolescents, including actions to reduce gender-based barriers taking into consideration sociodemographic characteristics of this population and the relationship they have with their partners.

Fatores associados ao acesso anterior à gestação a serviços de saúde por adolescentes gestantes / Factores asociados con el acceso anterior a la gestación a los servicios de salud por adolescentes gestantes / Factors associated with access to health services prior to pregnancy by pregnant adolescents

OBJETIVO: Analisar os fatores determinantes do acesso de adolescentes gestantes a serviços de atenção primária à saúde, anterior à ocorrência da gestação. MÉTODOS: Estudo transversal baseado em referencial teórico. O acesso a serviços foi analisado em cinco dimensões: geográfico, econômico, administrativo, psicossocial e de informação. Participaram 200 adolescentes primigestas (10 a 19 anos) atendidas em uma unidade básica de saúde do município de Indaiatuba (SP), em 2003. Um questionário com perguntas abertas e fechadas referentes ao acesso ao último serviço de saúde utilizado, anterior à gestação, foi aplicado às participantes no momento de sua primeira consulta de pré-natal. Os dados foram analisados por meio do teste de qui-quadrado de Pearson ou exato de Fisher e por regressão logística múltipla, considerando as cinco dimensões de acesso. RESULTADOS: Mais da metade (63,7 por cento) das adolescentes utilizou algum serviço de saúde para consulta ginecológica...

OBJECTIVE: To assess determinants of access to primary care services prior to pregnancy by pregnant adolescents. METHODS: Theory-based cross-sectional study conducted to examine access to heath services at five dimensions: geographic, economic, administrative, psychosocial and information. There were included in the study 200 first-time pregnant adolescents (aged 10 to 19 years) who attended a primary care unit in the municipality of Indaiatuba, Southeastern Brazil, in 2003. Data was collected using a semi-structured questionnaire applied in face-to-face interviews conducted just before their first pre-natal care visit. Data analyses were performed through Person's chi-square or Fisher's exact test, and by a multivariate logistic regression model including all five dimensions of access. RESULTS: More than half (63.7 percent) of the adolescents utilized some gynecological care service. Information (43.8 percent) or psychosocial feelings of embarrassment or fear (37.0 percent)...

OBJETIVO: Analizar los factores determinantes del acceso de adolescentes gestantes a los servicios de atención primaria a la salud, anterior a la ocurrencia de la gestación. MÉTODOS: Se efectuó estudio transversal basado en referencial teórico. El acceso a servicios fue analizado en cinco dimensiones: geográfico, económico, administrativo, psicosocial y de información. Participaron 200 adolescentes primigestas (10 a 19 años) atendidas en una unidad básica de salud del municipio de Indaiatuba (Sureste de Brasil), en 2003. Se aplicó a las participantes en el momento de su primera consulta pre-natal un cuestionario con preguntas abiertas y cerradas referentes al acceso al último servicio de salud utilizado, anterior a la gestación. Los datos fueron analizados por medio de la prueba de Chi cuadrado de Pearson o exacto de Fischer y por regresión logística múltiple, considerando las cinco dimensiones del acceso...

[Knowledge of pregnant adolescents about reproductive anatomy and physiology in a municipality of Southern Brazil]. / Conhecimento de adolescentes grávidas sobre anatomia e fisiologia da reprodução.

OBJECTIVES: To study knowledge of some aspects of the female reproductive anatomy and physiology and their association with socio-demographic and reproductive "choices" of pregnant adolescents. METHODS: A cross-sectional study was performed with 200 first time pregnant adolescents who attended a public women's health clinic in the municipality of Indaiatuba, Sao Paulo, Brazil. During their first prenatal care visit, face-to-face interviews were conducted using a structured questionnaire and a three-dimensional handmade female model. Bivariate data analyses were performed using Pearson's Chi-square or Fisher's Exact test. Data were also analyzed using multivariate logistic regression models to test for associations of indicators of knowledge of female reproductive anatomy, physiology of female reproductive organs and physiology of reproduction with socio-demographic characteristics and reproductive "choices". RESULTS: The majority had little knowledge of anatomy (55.5%), with external organs more easily identified and placed than the internal; of physiology of reproductive organs (61.0%), and of physiology of reproduction (76.5%). Associations were found between knowledge and age of partner, couple difference of age, maintenance of the relationship with partner after pregnancy, religious affiliation, and level of education. No association was found between indicators of knowledge with use of contraceptives at first intercourse and with intention of having the baby at that time. CONCLUSION: This study addressed the complexity of the relationship between knowledge of reproductive anatomy and physiology and the theme of adolescent pregnancy, and emphasized the need for more contextualized approaches of programmatic contents on sexual education, in view of the intention to reduce early pregnancy.

Conhecimento de adolescentes grávidas sobre anatomia e fisiologia da reprodução / Knowledge of pregnant adolescents about reproductive anatomy and physiology in a municipality of Southern Brazil

OBJETIVOS: Verificar o conhecimento sobre alguns aspectos do aparelho genital feminino, da fisiologia da reprodução e sua associação com características sociodemográficas e "escolhas" reprodutivas em adolescentes gestantes. MÉTODOS: Realizou-se um estudo de corte transversal com 200 adolescentes primigestas, durante a primeira consulta de pré-natal no Ambulatório da Mulher de Indaiatuba, São Paulo. Foram feitas entrevistas face a face, com questionário estruturado e um modelo feminino tridimensional confeccionado artesanalmente para coleta dos dados. Os dados foram analisados utilizando-se os testes Qui-quadrado de Pearson, ou Exato de Fisher, e por regressões logísticas múltiplas, para verificar associações entre indicadores do conhecimento sobre anatomia dos órgãos genitais femininos, fisiologia dos órgãos e da reprodução com características sociodemográficas e "escolhas" reprodutivas. RESULTADOS: A maioria tinha conhecimento insatisfatório sobre anatomia (55,5 por cento), com os órgãos externos sendo identificados com maior facilidade e melhor localizados do que os internos; fisiologia dos órgãos (61 por cento); e aspectos fisiológicos da reprodução (76,5 por cento). Algumas associações significativas foram estabelecidas entre o conhecimento e a idade dos parceiros, diferença de idade do casal, manutenção do vinculo após ocorrência da gravidez, filiação religiosa e escolaridade da adolescente. Não houve associação entre os indicadores de conhecimento estudados com a utilização de método anticoncepcional na primeira relação sexual e a intenção de ter um filho naquele momento. CONCLUSÃO: Este estudo faz emergir a complexidade da relação entre o conhecimento sobre anatomia e fisiologia reprodutivas e a temática da gravidez na adolescência, evidenciando a necessidade de abordagens mais contextualizadas dos conteúdos de programas de educação sexual, quando seu foco for a redução da gravidez precoce.

OBJECTIVES: To study knowledge of some aspects of the female reproductive anatomy and physiology and their association with socio-demographic and reproductive "choices" of pregnant adolescents. METHODS: A cross-sectional study was performed with 200 first time pregnant adolescents who attended a public women's health clinic in the municipality of Indaiatuba, Sao Paulo, Brazil. During their first prenatal care visit, face-to-face interviews were conducted using a structured questionnaire and a three-dimensional handmade female model. Bivariate data analyses were performed using Person's Chi-square or Fisher Exact test. Data were also analyzed using multivariate logistic regression models to test for associations of indicators of knowledge of female reproductive anatomy, physiology of female reproductive organs and physiology of reproduction with socio-demographic characteristics and reproductive "choices". RESULTS: The majority had little knowledge of anatomy (55.5 percent), with external organs more easily identified and placed than the internal; of physiology of reproductive organs (61.0 percent), and of physiology of reproduction (76.5 percent). Associations were found between knowledge and age of partner, couple difference of age, maintenance of the relationship with partner after pregnancy, religious affiliation, and level of education. No association was found between indicators of knowledge with use of contraceptives at first intercourse and with intention of having the baby at that time. CONCLUSION: This study addressed the complexity of the relationship between knowledge of reproductive anatomy and physiology and the theme of adolescent pregnancy, and emphasized the need for more contextualized approaches of programmatic contents on sexual education, in view of the intention to reduce early pregnancy.

Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block.

The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

A marriage of convenience: beta-subunits and voltage-dependent K+ channels.

The movement of ions across cell membranes is essential for a wide variety of fundamental physiological processes, including secretion, muscle contraction, and neuronal excitation. This movement is possible because of the presence in the cell membrane of a class of integral membrane proteins dubbed ion channels. Ion channels, thanks to the presence of aqueous pores in their structure, catalyze the passage of ions across the otherwise ion-impermeable lipid bilayer. Ion conduction across ion channels is highly regulated, and in the case of voltage-dependent K(+) channels, the molecular foundations of the voltage-dependent conformational changes leading to the their open (conducting) configuration have provided most of the driving force for research in ion channel biophysics since the pioneering work of Hodgkin and Huxley (Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-544). The voltage-dependent K(+) channels are the prototypical voltage-gated channels and govern the resting membrane potential. They are responsible for returning the membrane potential to its resting state at the termination of each action potential in excitable membranes. The pore-forming subunits (alpha) of many voltage-dependent K(+) channels and modulatory beta-subunits exist in the membrane as one component of macromolecular complexes, able to integrate a myriad of cellular signals that regulate ion channel behavior. In this review, we have focused on the modulatory effects of beta-subunits on the voltage-dependent K(+) (Kv) channel and on the large conductance Ca(2+)- and voltage-dependent (BK(Ca)) channel.

Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel.

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. The most remarkable effects of beta1 and beta2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by alpha and beta1 or beta2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of beta1 but not beta2. Here we reveal the molecular regions in these beta subunits that determine their differential functional coupling with the pore-forming alpha-subunit. We made chimeric constructs between beta1 and beta2 subunits, and BK channels formed by alpha and chimeric beta subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the beta1 and beta2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these beta subunits. Moreover, the intracellular domains of beta1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the alpha-subunit to be the target of the modulation by the beta1-subunit.