Thyroid Hormone Upregulates Cav1.2 Channels in Cardiac Cells via the Downregulation of the Channels' ß4 Subunit.

Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavß4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavß4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavß4. Finally, we found that overexpression of Cavß4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavß4 subunit expression.

Blockade of CaV3 calcium channels and induction of G0/G1 cell cycle arrest in colon cancer cells by gossypol.

BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.

A new active packaging system based on chickpea-based edible coatings added with microcapsules of Cosmos sulphureus Cav. flower extract.

BACKGROUND: The Cosmos sulphureus Cav. plant is studied for its high polyphenolic content with antioxidant properties. Its flowers, rich in phenolic acids, flavonoids, and tannins, hold promise as antioxidants in food preservation. The inclusion of these compounds in chickpea-based coatings with a previously studied preservative effect would be an excellent option as a food preservation method and microencapsulation addresses challenges like dispersion and degradation of polyphenols in the coating. The objective of this research was to evaluate the in vitro antioxidant activity of Cosmos sulphureus leaves, seed, and flower extracts and explore the protective effects of chickpea-based coatings containing microcapsules of flower polyphenolic extract on the chemical quality of stored roasted sunflower seeds during storage. RESULTS: The ethanolic leaf extract exhibited the highest antiradical activity, followed by the aqueous flower extract. After a storage period of 15 days, at 40 °C, the chickpea-based coatings effectively delayed lipid oxidation in the roasted sunflowers seeds, and the inclusion of polyphenolic microcapsules with 0.01% extract (SMC 0.01%) in the coating significantly improved the protective effect. By day 15 of storage, SMC 0.01% showed comparable peroxide value, conjugated dienes, and linoleic acid content to samples containing the synthetic antioxidant BHT (butylated hydroxytoluene). Samples that only contained chickpea-based coating and coating with polyphenolic microcapsules with 0.005% extract exhibited significantly greater reduction in fatty acid content compared to the 0.01% SMC treatment. CONCLUSION: The chickpea-based coating with polyphenolic microcapsules demonstrated antioxidant activity akin to synthetic BHT, offering a promising biopackaging solution for lipid-rich foods like roasted sunflower seeds. © 2024 Society of Chemical Industry.

Pronociceptive role of spinal Cav2.3 (R-type) calcium channels in a mouse model of postoperative pain.

BACKGROUND: More than 80% of patients may experience acute pain after a surgical procedure, and this is often refractory to pharmacological intervention. The identification of new targets to treat postoperative pain is necessary. There is an association of polymorphisms in the Cav2.3 gene with postoperative pain and opioid consumption. Our study aimed to identify Cav2.3 as a potential target to treat postoperative pain and to reduce opioid-related side effects. EXPERIMENTAL APPROACH: A plantar incision model was established in adult male and female C57BL/6 mice. Cav2.3 expression was detected by qPCR and suppressed by siRNA treatment. The antinociceptive efficacy and safety of a Cav2.3 blocker-alone or together with morphine-was also assessed after surgery. KEY RESULTS: Paw incision in female and male mice caused acute nociception and increased Cav2.3 mRNA expression in the spinal cord but not in the incised tissue. Intrathecal treatment with siRNA against Cav2.3, but not with a scrambled siRNA, prevented the development of surgery-induced nociception in both male and female mice, with female mice experiencing long-lasting effects. High doses of i.t. SNX-482, a Cav2.3 channel blocker, or morphine injected alone, reversed postoperative nociception but also induced side effects. A combination of lower doses of morphine and SNX-482 mediated a long-lasting reversal of postsurgical pain in female and male mice. CONCLUSION: Our results demonstrate that Cav2.3 has a pronociceptive role in the induction of postoperative pain, indicating that it is a potential target for the development of therapeutic approaches for the treatment of postoperative pain.

Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway.

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein.

Larrea divaricata a native Argentine plant with potential activity against SARS-CoV-2? / ¿Larrea divaricata una planta nativa argentina con actividad potencial sobre SARS-CoV-2?

Larrea divaricata Cav. is an autochthonous South American plant popularly used in inflammatory and infectious diseases with reported anti - inflammatory, immunomodulatory, antimicrobial and antioxidant activities. Covid - 19 is an infection ca used by the severe acute respiratory syndrome coronavirus 2 (SARS - CoV - 2). This virus can cause pneumonia and even death in about 5% of the cases. The objective of the article was to demonstrate, through a literature review, that L. divaricata has sufficie nt attributes to be assayed against SARS - CoV - 2. For this, the chemical composition, reported activities and docking studies were taken into account. This review demonstrated that the plant extracts are capable of inhibiting the proliferation of fungi, bact eria and viruses and that they exert anti - inflammatory and immunomodulatory actions in different " in vitro " and " in vivo " models. These results suggest that the plant is a good candidate to be studied for the prevention and/or treatment of SARS - CoV - 2.

Larrea divaricata Cav. es una planta autóctona Sudamericana, utilizada popularmente en enfermedades inflamatorias e infecciosas, con activida d anti - inflamatoria, inmunomoduladora, antimicrobiana y antioxidante reportada. El Covid - 19 es una infección causada por una cepa de coronavirus, SARS - CoV - 2 (coronavirus tipo 2 causante del síndrome respiratorio agudo severo). Este virus puede originar neu monía e incluso la muerte en alrededor del 5% de los casos. Nuestro objetivo fue demostrar, a través de una revisión bibliográfica, que esta planta tiene atributos suficientes para ser ensayada en estudios contra SARS - CoV - 2. Se tuvo en cuenta la composici ón química, los antecedentes científicos y los estudios de acoplamiento molecular. Esta revisión permitió demostrar que extractos de la planta son capaces de inhibir la proliferación de hongos, bacterias y virus y que presentan acción anti - inflamatoria en diferentes modelos " in vitro " e " in vivo ", lo que los hace candidatos a ser estudiados en la prevención y/o tratamiento de la infección contra SARS - CoV - 2.

ω-Phonetoxins inhibit voltage-gated calcium CaV2.2 ion channel splice isoforms of dorsal root ganglia.

Cell-specific alternative splicing of Cacna1b pre-mRNA generates functionally distinct voltage-gated CaV2.2 channels. CaV2.2 channels mediate the release of glutamate from nociceptor termini in the dorsal horn spinal cord and they are implicated in chronic pain. One alternatively spliced exon in Cacna1b, e37a, is highly expressed in dorsal root ganglia, relative to other regions of the nervous system, and it is particularly important in inflammatory hyperalgesia. Here we studied the effects of two ω-phonetoxins, PnTx3-4 and Phα1ß, derived from the spider Phoneutria nigriventer on CaV2.2 channel isoforms of dorsal root ganglia (CaV2.2 e37a and CaV2.2 e37b). Both PnTx3-4 and Phα1ß are known to have analgesic effects in rodent models of pain and to inhibit CaV2.2 channels. CaV2.2 e37a and CaV2.2 e37b isoforms expressed in a mammalian cell line were inhibited by PnTx3-4 and Phα1ß with similar potency and with similar timecourse, although CaV2.2 e37a currents were slightly, but consistently more sensitive to toxin inhibition compared to CaV2.2 e37b. The inhibitory effects of PnTx3-4 and Phα1ß on CaV2.2-e37a and CaV2.2-e37b channels were voltage-dependent, and both occlude the inhibitory effects of ω-conotoxin GVIA, consistent with a common site of action. The potency of PnTx3-4 and Phα1ß on both major splice isoforms in dorsal root ganglia constribute to understanding the analgesic actions of these ω-phonetoxins.

T-type calcium channel modulation by hydrogen sulfide in neuropathic pain conditions.

Neuropathic pain can appear as a direct or indirect nerve damage lesion or disease that affects the somatosensory nervous system. If the neurons are damaged or indirectly stimulated, immune cells contribute significantly to inflammatory and neuropathic pain. After nerve injury, peripheral macrophages/spinal microglia accumulate around damaged neurons, producing endogenous hydrogen sulfide (H2S) through the cystathionine-γ-lyase (CSE) enzyme. H2S has a pronociceptive modulation on the Cav3.2 subtype, the predominant Cav3 isoform involved in pain processes. The present review provides relevant information about H2S modulation on the Cav3.2 T-type channels in neuropathic pain conditions. We have discussed that the dual effect of H2S on T-type channels is concentration-dependent, that is, an inhibitory effect is seen at low concentrations of 10 µM and an augmentation effect on T-current at 100 µM. The modulation mechanism of the Cav3.2 channel by H2S involves the direct participation of the redox/Zn2+ affinity site located in the His191 in the extracellular loop of domain I of the channel, involving a group of extracellular cysteines, comprising C114, C123, C128, and C1333, that can modify the local redox environment. The indirect interaction pathways involve the regulation of the Cav3.2 channel through cytokines, kinases, and post-translational regulators of channel expression. The findings conclude that the CSE/H2S/Cav3.2 pathway could be a promising therapeutic target for neuropathic pain disorders.

Role of Cav2.3 (R-type) Calcium Channel in Pain and Analgesia: A Scoping Review.

BACKGROUND: Voltage-gated calcium channels (VGCCs) play an important role in pain development and maintenance. As Cav2.2 and Cav3.2 channels have been identified as potential drug targets for analgesics, the participation of Cav2.3 (that gives rise to R-type calcium currents) in pain and analgesia remains incompletely understood. OBJECTIVE: Identify the participation of Cav2.3 in pain and analgesia. METHODS: To map research in this area as well as to identify any existing gaps in knowledge on the potential role of Cav2.3 in pain signalling, we conducted this scoping review. We searched PubMed and SCOPUS databases, and 40 articles were included in this study. Besides, we organized the studies into 5 types of categories within the broader context of the role of Cav2.3 in pain and analgesia. RESULTS: Some studies revealed the expression of Cav2.3 in pain pathways, especially in nociceptive neurons at the sensory ganglia. Other studies demonstrated that Cav2.3-mediated currents could be in-hibited by analgesic/antinociceptive drugs either indirectly or directly. Some articles indicated that Cav2.3 modulates nociceptive transmission, especially at the pre-synaptic level at spinal sites. There are studies using different rodent pain models and approaches to reduce Cav2.3 activity or expression and mostly demonstrated a pro-nociceptive role of Cav2.3, despite some contradictory findings and deficiencies in the description of study design quality. There are three studies that reported the association of single-nucleotide polymorphisms in the Cav2.3 gene (CACNA1E) with postoperative pain and opioid consumption as well as with the prevalence of migraine in patients. CONCLUSION: Cav2.3 is a target for some analgesic drugs and has a pro-nociceptive role in pain.

Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels.

The present study was undertaken to explore the relative contributions of Cav3.2 T-type channels to mediating the antihyperalgesic activity of joint manipulation (JM) therapy. We used the chronic constriction injury model (CCI) to induce peripheral neuropathy and chronic pain in male mice, followed by JM. We demonstrate that JM produces long-lasting mechanical anti-hyperalgesia that is abolished in Cav3.2 null mice. Moreover, we found that JM displays a similar analgesic profile as the fatty acid amide hydrolase inhibitor URB597, suggesting a possible converging mechanism of action involving endocannabinoids. Overall, our findings advance our understanding of the mechanisms through which JM produces analgesia.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines.

INTRODUCTION: Cholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. MATERIALS AND METHODS: Cell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-ß-cyclodextrin (MßCD - 7.5, 10 or 15 mM) RESULTS: Decreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein ß-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. CONCLUSION: Cholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

Constitutive activity of the dopamine (D5 ) receptor, highly expressed in CA1 hippocampal neurons, selectively reduces CaV 3.2 and CaV 3.3 currents.

BACKGROUND AND PURPOSE: CaV 3.1-3 currents differentially contribute to neuronal firing patterns. CaV 3 are regulated by G protein-coupled receptors (GPCRs) activity, but information about CaV 3 as targets of the constitutive activity of GPCRs is scarce. We investigate the impact of D5 recpetor constitutive activity, a GPCR with high levels of basal activity, on CaV 3 functionality. D5 recpetor and CaV 3 are expressed in the hippocampus and have been independently linked to pathophysiological states associated with epilepsy. EXPERIMENTAL APPROACH: Our study models were HEK293T cells heterologously expressing D1 or D5 receptor and CaV 3.1-3, and mouse brain slices containing the hippocampus. We used chlorpromazine (D1 /D5 inverse agonist) and a D5 receptor mutant lacking constitutive activity as experimental tools. We measured CaV 3 currents and excitability parameters using the patch-clamp technique. We completed our study with computational modelling and imaging technique. KEY RESULTS: We found a higher sensitivity to TTA-P2 (CaV 3 blocker) in CA1 pyramidal neurons obtained from chlorpromazine-treated animals compared with vehicle-treated animals. We found that CaV 3.2 and CaV 3.3-but not CaV 3.1-are targets of D5 receptor constitutive activity in HEK293T cells. Finally, we found an increased firing rate in CA1 pyramidal neurons from chlorpromazine-treated animals in comparison with vehicle-treated animals. Similar changes in firing rate were observed on a neuronal model with controlled CaV 3 currents levels. CONCLUSIONS AND IMPLICATIONS: Native hippocampal CaV 3 and recombinant CaV 3.2-3 are sensitive to D5 receptor constitutive activity. Manipulation of D5 receptor constitutive activity could be a valuable strategy to control neuronal excitability, especially in exacerbated conditions such as epilepsy.

Epileptic Targets and Drugs: A Mini-Review.

BACKGROUND: Epilepsy is a neurological disease affected by an imbalance of inhibitory and excitatory signaling in the brain. INTRODUCTION: In this disease, the targets are active in pathophysiology and thus can be used as a focus for pharmacological treatment. METHODS: Several studies demonstrated the antiepileptic effect of drugs acting on the following targets: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-gated calcium channel (Cav), Gamma aminobutyric acid transporter type 1 (GAT1), voltage-gated sodium channels (Nav), voltage-gated potassium channel of the Q subfamily (KCNQ) and Gamma aminobutyric acid type A (GABAA) receiver. RESULTS: These studies highlight the importance of molecular docking. CONCLUSION: Quantitative Structure-Activity Relationship (QSAR) and computer aided drug design (CADD) in predicting of possible pharmacological activities of these targets.

Fowl Aviadenovirus E associated with hepatitis-hydropericardium syndrome in broiler breeders / Síndrome da hepatite-hidropericárdio por Aviadenovirus e em reprodutores de corte

ABSTRACT: Fowl aviadenovirus (FAdV) is an important pathogen in the global poultry industry and the etiology of inclusion body hepatitis-hydropericardium syndrome (HHS) in chickens. Since the 1990s, several outbreaks of HHS have occurred in poultry producing areas, including South America. The coinfection of FAdV and chicken anemia virus (CAV) may markedly impact the incidence of HHS. This study describes an outbreak of HHS in coinfection with CAV in industrial broiler breeders and characterizes the FAdV isolate. The three-week-old male broiler breeders had pale bone marrow, enlarged and yellowish liver, splenomegaly, and atrophied thymus; one chicken was also found with hydropericardium. Virus isolation was performed in SPF chicken embryos of liver and thymus. Tissues of the naturally infected chickens and the inoculated embryos were evaluated by PCR and histopathology. All affected chickens and inoculated embryos were positive for FAdV and CAV. The inoculated embryos had enlarged, greenish and hemorrhagic livers, and 30% died within 7 days of inoculation. Phylogenetic analysis of the FAdV isolate hexon gene partial sequence enabled grouping with E species. The E species has recently become a relevant species in several countries. The association of FAdV with CAV in breeders is of further concern due to both being capable of vertical transmission. Within the last decade, a worldwide upsurge of HHS in broiler breeders owing to failed biosecurity has occurred. In this episode, the failure on biosecurity may have enabled challenge with both FAdV and CAV, with pathological synergism. The CAV-impaired adaptive immunity may have benefited the FAdV infection.

RESUMO: Adenovírus aviário (FAdV) é um importante patógeno na indústria avícola global e a etiologia da síndrome da hepatite por corpúsculo de inclusão-hidropericárdio (SHH) em galinhas. Desde a década de 1990, vários surtos de SHH foram descritos em todas as áreas de produção de aves, incluindo na América do Sul, e a coinfecção entre FAdV e vírus da anemia das galinhas (CAV) pode ser agravante para todos os aspectos da SHH. Objetiva-se descrever um surto de SHH em matrizes de frangos corte, caracterizar a estirpe de FAdV envolvida e destacar a coinfecção com CAV. Foram avaliados machos reprodutores de corte com três semanas de idade, com medula óssea pálida, fígado aumentado e amarelado e esplenomegalia, timo atrofiado e um com hidropericárdio. Fígado e timo foram coletados para isolamento do vírus em embriões de galinhas SPF, PCR e histopatologia. Todas as aves acometidas e embriões inoculados foram positivos para FAdV e CAV. Os embriões inoculados tiveram óbito de 30% em até sete dias após a inoculação e alterações hepáticas por fígados esverdeados e aumentados. A análise filogenética de FAdV com base em parte da sequência do gene que codifica a proteína hexon revelou identidade com a espécie E, que se tornou disseminada em vários países. A coinfecção de FAdV e CAV resulta em maior intensidade de lesões, maior morbidade e mortalidade e em reprodutores tem alta relevância epidemiológica, em razão da transmissão vertical de ambos e da ampla distribuição geográfica das progênies infectadas. Na última década, ocorreu um aumento mundial na ocorrência de SHH em frangos de corte relacionado a falhas em biosseguridade, especialmente em reprodutores, condição que pode ter ocorrido neste episódio. A presença de FAdV e CAV em reprodutores é motivo para preocupação por reflexos negativos à imunidade e viabilidade das progênies.

Fowl Aviadenovirus E associated with hepatitis-hydropericardium syndrome in broiler breeders / Síndrome da hepatite-hidropericárdio por Aviadenovirus e em reprodutores de corte

Fowl aviadenovirus (FAdV) is an important pathogen in the global poultry industry and the etiology of inclusion body hepatitis-hydropericardium syndrome (HHS) in chickens. Since the 1990s, several outbreaks of HHS have occurred in poultry producing areas, including South America. The coinfection of FAdV and chicken anemia virus (CAV) may markedly impact the incidence of HHS. This study describes an outbreak of HHS in coinfection with CAV in industrial broiler breeders and characterizes the FAdV isolate. The three-week-old male broiler breeders had pale bone marrow, enlarged and yellowish liver, splenomegaly, and atrophied thymus; one chicken was also found with hydropericardium. Virus isolation was performed in SPF chicken embryos of liver and thymus. Tissues of the naturally infected chickens and the inoculated embryos were evaluated by PCR and histopathology. All affected chickens and inoculated embryos were positive for FAdV and CAV. The inoculated embryos had enlarged, greenish and hemorrhagic livers, and 30% died within 7 days of inoculation. Phylogenetic analysis of the FAdV isolate hexon gene partial sequence enabled grouping with E species. The E species has recently become a relevant species in several countries. The association of FAdV with CAV in breeders is of further concern due to both being capable of vertical transmission. Within the last decade, a worldwide upsurge of HHS in broiler breeders owing to failed biosecurity has occurred. In this episode, the failure on biosecurity may have enabled challenge with both FAdV and CAV, with pathological synergism. The CAV-impaired adaptive immunity may have benefited the FAdV infection.

Adenovírus aviário (FAdV) é um importante patógeno na indústria avícola global e a etiologia da síndrome da hepatite por corpúsculo de inclusão-hidropericárdio (SHH) em galinhas. Desde a década de 1990, vários surtos de SHH foram descritos em todas as áreas de produção de aves, incluindo na América do Sul, e a coinfecção entre FAdV e vírus da anemia das galinhas (CAV) pode ser agravante para todos os aspectos da SHH. Objetiva-se descrever um surto de SHH em matrizes de frangos corte, caracterizar a estirpe de FAdV envolvida e destacar a coinfecção com CAV. Foram avaliados machos reprodutores de corte com três semanas de idade, com medula óssea pálida, fígado aumentado e amarelado e esplenomegalia, timo atrofiado e um com hidropericárdio. Fígado e timo foram coletados para isolamento do vírus em embriões de galinhas SPF, PCR e histopatologia. Todas as aves acometidas e embriões inoculados foram positivos para FAdV e CAV. Os embriões inoculados tiveram óbito de 30% em até sete dias após a inoculação e alterações hepáticas por fígados esverdeados e aumentados. A análise filogenética de FAdV com base em parte da sequência do gene que codifica a proteína hexon revelou identidade com a espécie E, que se tornou disseminada em vários países. A coinfecção de FAdV e CAV resulta em maior intensidade de lesões, maior morbidade e mortalidade e em reprodutores tem alta relevância epidemiológica, em razão da transmissão vertical de ambos e da ampla distribuição geográfica das progênies infectadas. Na última década, ocorreu um aumento mundial na ocorrência de SHH em frangos de corte relacionado a falhas em biosseguridade, especialmente em reprodutores, condição que pode ter ocorrido neste episódio. A presença de FAdV e CAV em reprodutores é motivo para preocupação por reflexos negativos à imunidade e viabilidade das progênies.

The ubiquitin E3 ligase Parkin regulates neuronal CaV1.3 channel functional expression.

Neuronal L-type Ca2+ channels of the CaV1.3 subclass are transmembrane protein complexes that contribute to the pacemaker activity in the adult substantia nigra dopaminergic neurons. The altered function of these channels may play a role in the development and progress of neurodegenerative mechanisms implicated in Parkinson's disease (PD). Although L-type channel expression is precisely regulated, an increased functional expression has been observed in PD. Previously, we showed that Parkin, an E3 enzyme of the ubiquitin-proteasome system (UPS) interacts with neuronal CaV2.2 channels promoting their ubiquitin-mediated degradation. In addition, previous studies show an increase in CaV1.3 channel activity in dopaminergic neurons of the SNc and that Parkin expression is reduced in PD. These findings suggest that the decrease in Parkin may affect the proteasomal degradation of CaV1.3, which helps explain the increase in channel activity. Therefore, the present report aims to gain insight into the degradation mechanisms of the neuronal CaV1.3 channel by the UPS. Immunoprecipitation assays showed the interaction between Parkin and the CaV1.3 channels expressed in HEK-293 cells and neural tissues. Likewise, Parkin overexpression reduced the total and membrane channel levels and decreased the current density. Consistent with this, patch-clamp recordings in the presence of an inhibitor of the UPS, MG132, prevented the effects of Parkin, suggesting enhanced channel proteasomal degradation. In addition, the half-life of the pore-forming CaV1.3α1 protein was significantly reduced by Parkin overexpression. Finally, electrophysiological recordings using a PRKN knockout HEK-293 cell line generated by CRISPR/Cas9 showed increased current density. These results suggest that Parkin promotes the proteasomal degradation of CaV1.3, which may be a relevant aspect for the pathophysiology of PD.NEW & NOTEWORTHY The increased expression of CaV1.3 calcium channels is a crucial feature of Parkinson's disease (PD) pathophysiology. However, the mechanisms that determine this increase are not yet defined. Parkin, an enzyme of the ubiquitin-proteasome system, is known to interact with neuronal channels promoting their ubiquitin-mediated degradation. Interestingly, Parkin mutations also play a role in PD. Here, the degradation mechanisms of CaV1.3 channels and their relationship with the pathophysiology of PD are studied in detail.

The role of voltage-gated calcium channels in the pathogenesis of Parkinson's disease.

Aim: Voltage-gated calcium (CaV) channels play an essential role in maintaining calcium homeostasis and regulating numerous physiological processes in neurons. Therefore, dysregulation of calcium signaling is relevant in many neurological disorders, including Parkinson's disease (PD). This review aims to introduce the role of CaV channels in PD and discuss some novel aspects of channel regulation and its impact on the molecular pathophysiology of the disease.Methods: an exhaustive search of the literature in the field was carried out using the PubMed database of The National Center for Biotechnology Information. Systematic searches were performed from the initial date of publication to May 2022.Results: Although α-synuclein aggregates are the main feature of PD, L-type calcium (CaV1) channels seem to play an essential role in the pathogenesis of PD. Changes in the functional expression of CaV1.3 channels alter Calcium homeostasis and contribute to the degeneration of dopaminergic neurons. Furthermore, recent studies suggest that CaV channel trafficking towards the cell membrane depends on the activity of the ubiquitin-proteasome system (UPS). In PD, there is an increase in the expression of L-type channels associated with a decrease in the expression of Parkin, an E3 enzyme of the UPS. Therefore, a link between Parkin and CaV channels could play a fundamental role in the pathogenesis of PD and, as such, could be a potentially attractive target for therapeutic intervention.Conclusion: The study of alterations in the functional expression of CaV channels will provide a framework to understand better the neurodegenerative processes that occur in PD and a possible path toward identifying new therapeutic targets to treat this condition.

Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca2+ Pump Upregulation Counterbalances Cav1.2-Mediated Ca2+ Influx in Mesenteric Arteries.

In mesenteric arteries (MAs), aldosterone (ALDO) binds to the endogenous mineralocorticoid receptor (MR) and increases the expression of the voltage-gated L-type Cav1.2 channel, an essential ion channel for vascular contraction, sarcoplasmic reticulum (SR) Ca2+ store refilling, and Ca2+ spark generation. In mesenteric artery smooth muscle cells (MASMCs), Ca2+ influx through Cav1.2 is the indirect mechanism for triggering Ca2+ sparks. This process is facilitated by plasma membrane-sarcoplasmic reticulum (PM-SR) nanojunctions that drive Ca2+ from the extracellular space into the SR via Sarco/Endoplasmic Reticulum Ca2+ (SERCA) pump. Ca2+ sparks produced by clusters of Ryanodine receptors (RyRs) at PM-SR nanodomains, decrease contractility by activating large-conductance Ca2+-activated K+ channels (BKCa channels), which generate spontaneous transient outward currents (STOCs). Altogether, Cav1.2, SERCA pump, RyRs, and BKCa channels work as a functional unit at the PM-SR nanodomain, regulating intracellular Ca2+ and vascular function. However, the effect of the ALDO/MR signaling pathway on this functional unit has not been completely explored. Our results show that short-term exposure to ALDO (10 nM, 24 h) increased the expression of Cav1.2 in rat MAs. The depolarization-induced Ca2+ entry increased SR Ca2+ load, and the frequencies of both Ca2+ sparks and STOCs, while [Ca2+]cyt and vasoconstriction remained unaltered in Aldo-treated MAs. ALDO treatment significantly increased the mRNA and protein expression levels of the SERCA pump, which counterbalanced the augmented Cav1.2-mediated Ca2+ influx at the PM-SR nanodomain, increasing SR Ca2+ content, Ca2+ spark and STOC frequencies, and opposing to hyperpolarization-induced vasoconstriction while enhancing Acetylcholine-mediated vasorelaxation. This work provides novel evidence for short-term ALDO-induced upregulation of the functional unit comprising Cav1.2, SERCA2 pump, RyRs, and BKCa channels; in which the SERCA pump buffers ALDO-induced upregulation of Ca2+ entry at the superficial SR-PM nanodomain of MASMCs, preventing ALDO-triggered depolarization-induced vasoconstriction and enhancing vasodilation. Pathological conditions that lead to SERCA pump downregulation, for instance, chronic exposure to ALDO, might favor the development of ALDO/MR-mediated augmented vasoconstriction of mesenteric arteries.

Direct inhibition of CaV2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction.

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel ß subunit (CaVß), and the binding between CaVα1/CaVß appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVß. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVß independent promotion of dynamin-dependent endocytosis.