Ischemic cerebrovascular events are among the leading causes of death and disability worldwide, disproportionately affecting the elderly due to vascular aging, oxidative stress, and chronic inflammation. During cerebral ischemia, oxygen and glucose deprivation trigger a cascade of pathological events, including excitotoxicity, mitochondrial dysfunction, blood-brain barrier disruption, and neuroinflammation. Emerging evidence identifies Klotho as a multifunctional protein with anti-aging, antioxidant, and anti-inflammatory properties that may confer neuroprotection against ischemic injury. Aging is associated with a progressive decline in Klotho expression, correlating with increased stroke susceptibility and poorer recovery outcomes. Experimental studies demonstrate that Klotho attenuates glutamatergic excitotoxicity by regulating the GluN2B subunit and excitatory amino acid transporters. It reduces oxidative stress through inhibition of NADPH oxidase and activation of SOD2 and FOXO3 pathways, while preserving blood-brain barrier integrity by downregulating aquaporin-4 and matrix metalloproteinase-9. Moreover, Klotho suppresses pro-inflammatory signaling by inhibiting NF-κB, RelA, and NLRP3 inflammasome activation, and enhances anti-inflammatory responses via PPAR-γ modulation. Beyond limiting neuronal death, Klotho promotes neurogenesis and synaptic plasticity by regulating neurotrophic factors and Wnt signaling, thereby supporting neural stem cell survival and functional recovery after ischemia. Collectively, these findings position Klotho as a central regulator of neuronal homeostasis and post-ischemic repair. This review provides the first comprehensive mechanistic integration of Klotho's neuroprotective role in ischemic cerebrovascular injury, proposing it as a unifying molecular axis linking brain aging to ischemic vulnerability and regenerative capacity.

Background and Aim The intranasal vaccine HB-ATV-8 contains the Seq-1 peptide (CHLLVDFLQSLS) as the active component within a nanoparticle formulation. It targets the cholesterol ester transfer protein (CETP) by inducing the formation of specific autoantibodies and a cellular antifibrotic response. It has demonstrated efficacy in preventing atherogenesis, liver steatosis, and fibrosis in preclinical models. Given that previously synthesized CETP inhibitors have been associated with increased cardiovascular risk, a preclinical safety assessment of HB-ATV-8 was conducted. Results Histopathological analysis of cardiac tissue from control and high-fat diet-fed rabbits treated with HB-ATV-8 revealed no inflammatory damage attributable to the vaccine. To further assess cardiac safety, an in vitro model using cells transfected with the human ether-à-go-go-related gene (hERG) potassium channel was employed to evaluate proarrhythmic potential through cardiac repolarization assessment. Patch-clamp electrophysiology confirmed that the HB-ATV-8 vaccine had no adverse effects on hERG channel kinetics. Genotoxicity was evaluated using the Ames test, which detects point mutations caused by base pair substitutions, additions, or deletions in DNA. The test was performed across multiple Salmonella typhimurium strains. The assays confirmed that HB-ATV-8 is not mutagenic. In addition, positron emission tomography (PET) imaging was performed in dwarf rabbits to evaluate glucose metabolism and systemic effects during vaccine treatment. PET imaging demonstrated that the HB-ATV-8 vaccine does not induce metabolic alterations and provided insights into its biodistribution. Conclusion Our data fully justify advancing the HB-ATV-8 vaccine into clinical development, where its potential to address both atherosclerosis and metabolic dysfunction-associated fatty liver disease (MAFLD) will be evaluated in human studies.

OBJECTIVE:Cerebral edema is a major contributor to mortality in diverse pathologies; regretfully, no effective therapies can reduce the associated damage. In cerebral ischemia, ionic edema formation is mediated by the sulfonylurea receptor 1 (SUR1)/transient receptor potential melastatin 4 (TRPM4) complex expressed in brain endothelial cells. Given that Resveratrol reduces the damage induced in human brain endothelial cells (HBEC-5i) subjected to oxygen-glucose deprivation (OGD)/reoxiygenation (R) by down-regulating SUR1 expression, we aimed to determine whether Resveratrol modulates SUR1-TRPM4 activity in this model. METHODS:HBEC-5i cells were exposed to OGD for 2 h followed by 24 h of R. Intracellular Na+ accumulation, measured with the Na-sensitive fluorescence probe coronaNa Green, was used as an indicator of SUR1-TRPM4 activity. Immunofluorescence assays were performed to evaluate SUR1 and TRPM4 expression. Resveratrol (5 μM) was administered during OGD/R. Diazoxide was used to identify responding cells. RESULTS:Resveratrol reduced the increase in intracellular Na+ induced by OGD/R and linked to SUR1/TRPM4 activation. While the basal SUR1 and TRPM4 expression levels were low, OGD/R induced their overexpression. Nonetheless, their intracellular distribution indicated incomplete co-localization. A subset of cells showed Na+ elevation after OGD/R, suggesting that although many cells expressed SUR1 and TRPM4, most did not form functional complexes. Diazoxide increased Na+ influx exclusively in 'responding' cells, confirming functional SUR1-TRPM4 activity in this subpopulation. CONCLUSION:SUR1-TRPM4 becomes functionally expressed in a subset of HBEC-5i during OGD/R and pretreatment with Resveratrol attenuated this response. Early modulation of SUR1-TRPM4 by Resveratrol may represent a potential strategy to limit ionic edema.

Anterior segment dysgeneses (ASDs) are a heterogeneous group of ocular developmental anomalies commonly associated with severe visual disability in pediatric age. Here, we report the generation of the iPSC line IOCVi002-A from a patient with a homozygous pathogenic c.292 T > C (p.(Y98H)) variant in the FOXE3 gene causing an ASD phenotype characterized by sclerocornea and aphakia. IOCVi002-A cells shows normal morphology, typical stemness and pluripotency. This iPSC line can be used for in vitro disease modeling for developmental ocular anomalies affecting anterior structures of the eye.

After ejaculation, mammalian spermatozoa are not capable of fertilizing a metaphase II-arrested egg. They require to undergo a series of biochemical and physiological processes collectively known as capacitation. In all these processes, the regulation of calcium ions fluxes plays essential roles and involves participation of many channels and transporters localized in the plasma membrane as well as in the membrane of intracellular organelles. In mammalian sperm, a fraction of these molecules has been proposed to contribute to mature sperm function. However, in many cases, the evidence for the presence of a given protein is based on the use of agonists and antagonists with more than one target. In this review, we will critically analyze the published evidence supporting the presence of these molecules in mammalian sperm with special emphasis to methods involving tandem mass spectrometry identification, electrophysiological evidence and controlled immunoassays.

Accurate determinations of acrosome reaction (AR) are fundamental to obtain physiologically relevant information of this essential event for sperm fertilization. For decades, the AR was exclusively studied in fixed preparation; more recently, new methods were developed to evaluate the AR in live sperm and in real time. These new technologies involved the use of genetically modified mice models, dyes with ion affinity, and fluorescent microscopy. In addition, these techniques allowed the reproduction field the possibility to follow the AR directly in the female tract under physiological conditions. Despite these advances, maintaining transgenic colonies is expensive for most research laboratories. Here, we present a methodology which can be mounted in a simple epifluorescence microscopy set in combination with appropriate filters enabling to follow the AR in real time. The implemented technique allows also to follow the AR in combination with [Ca2+]i, an ion directly involved in the regulation of this exocytotic reaction. In this chapter, we present in a simple way the methodology required to obtain AR/[Ca2+]i determinations using Fluo-4 and FM4-64 fluorescence recordings in live sperm under physiological and non-physiological stimuli. Also, we explain step by step the analysis and treatment to the images obtained during the recordings to determine AR quantitative information. The general methodology can be applied to measure other sperm parameters for which fluorescent dyes are available, such as intracellular pH (pHi) in combination with [Ca2+]i and/or the AR.

The sulfonylurea receptor 1 (SUR1) has been classified as a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily. SUR1, unlike the classic ABC transporters, assembles with Kir6.2, forming KATP channels to regulate the flux of potassium ions. In the central nervous system, SUR1 is weakly expressed in some brain regions but is induced by pathological conditions in the different cell types of the neurovascular unit. Therefore, we first analyzed the expression of SUR1 in various rat tissues and brain regions to identify SUR1 isoforms and their mRNA exon composition under physiological conditions. Later, we focused on the SUR1 expression in the brain and heart after ischemia/reperfusion. We observed two SUR1 isoforms (170 and 60-75 kDa) abundantly expressed in most rat tissues, except for the testis and brain, where basal expression of these isoforms was relatively low and exhibit a band of 100 kDa. Every exons coding for the functional domains of SUR1 mRNA were amplified from the tissues and brain regions analyzed. Results from in vitro and in vivo experiments indicated that SUR1 isoforms previously identified (170 and 60-75 kDa) were dramatically overexpressed in the brain after middle cerebral artery occlusion followed by reperfusion. In contrast, myocardial infarction followed by reperfusion significantly reduced SUR1 isoform expression in the heart. This study demonstrates the expression of at least two SUR1 isoforms in various tissues and suggests that ischemic processes may differentially regulate SUR1 expression depending on the tissue injured.

Drug repurposing is an increasingly recognized strategy in pharmaceutical development that focuses on identifying new therapeutic uses for already approved drugs, as an alternative to the time-consuming and costly process of developing new molecular entities. This approach has gained traction in oncology, especially in exploring the anticancer potential of non-oncologic agents. In recent years, the antipsychotic drug penfluridol (PNFL) has been identified as having antitumoral properties; however, the underlying mechanisms remain poorly understood. We hypothesized that PNFL may exert its effects by inhibiting the oncogenic potassium channel Kv10.1. Our results demonstrate that PNFL inhibits Kv10.1 activity and reduces cell migration in HEK-Kv10.1 cells. These findings suggest a novel mechanism that may contribute to the drug’s antitumoral effects.

Platelet hyperreactivity is a risk factor for cardiovascular disease development and mortality in hypertensive patients. Although several studies have linked platelet hyperreactivity with increased bone marrow thrombopoiesis, extramedullary thrombopoiesis remains highly unexplored in hypertension and other cardiovascular diseases. Here, we investigated thrombopoiesis-related parameters in the bone marrow, spleen, and lungs of 5-week-old spontaneously hypertensive rats (SHR) and their normotensive counterparts, Wistar Kyoto rats by quantitative histological imaging, flow cytometry, and enzyme-linked immunoassays. Flow cytometry assays and quantitative histological imaging strongly suggest that thrombocytosis in SHR is caused solely by bone marrow thrombopoiesis. Moreover, analysis of the lungs showed a significant decrease in the density of megakaryocyte and CKITpos/CD41pos cells, suggesting potential implications for lung megakaryopoiesis of SHR rats. Finally, while IL6 levels increased by twofold in the bone marrow, TPO levels decreased in both the bone marrow and serum of SHR rats. Overall, our results raise the question of whether pulmonary and bone marrow thrombopoiesis may regulate each other through TPO-based feedback, posing key questions about platelet biology under steady state and pathological conditions.

Un funcionamiento cerebral adecuado requiere de un suministro constante de sustratos energéticos, principalmente de glucosa, los cuales son transportados en la sangre circulando por las arterias que recorren todo el organismo. En el cerebro, estas arterias controlan rigurosamente el transporte de todas las moléculas que llegan al cerebro, y están constituidas por el endotelio vascular cerebral, que forma la barrera hematoencefálica. En un infarto cerebral, el flujo sanguíneo se ve interrumpido y el suministro de los compuestos vitales disminuye, lo que provoca una alteración en las funciones cerebrales; entonces el individuo sufrirá fallas en el movimiento, el lenguaje o en alguna de las funciones controlada en la región afectada del cerebro. Además, mientras no se recupere el flujo sanguíneo se activarán muchos procesos de daño, entre éstos destaca uno muy peligroso que puede causar la muerte: el incremento en la cantidad de agua contenida en el cerebro, conocido como edema. Su formación puede provocar una catástrofe como la vivida por los pasajeros del Titanic, quienes nunca sospecharon que el gran coloso pudiera ser vulnerable y naufragar. En este artículo, relataremos la forma en que un infarto cerebral puede provocar la acumulación de agua en este tejido.

Este artículo aborda de manera sencilla la estructura y función de la barrera hematoencefálica, destacando que a pesar de ser una barrera defensiva que protege el cerebro, no es estática; por lo contrario, es altamente dinámica y capaz de regular el tráfico de moléculas entre el flujo sanguíneo y el territorio cerebral. Al comparar las asombrosas similitudes que comparten las barreras naturales en el cuerpo humano con las fronteras físicas utilizadas por la humanidad para delimitar o proteger territorios, se hace hincapié en lo fundamental de su papel en el correcto funcionamiento del cerebro. Del mismo modo, se aborda el impacto que tiene el daño a esta estructura defensiva en el desarrollo de neuropatologías.

The SUR1-TRPM4-AQP4 complex is overexpressed in the initial phase of edema induced after cerebral ischemia, allowing the massive internalization of Na + and water within the brain micro endothelial cells (BMEC) of the blood-brain barrier. The expression of the Abcc8 gene encoding SUR1 depends on transcriptional factors that are responsive to oxidative stress. Because reactive oxygen species (ROS) are generated during cerebral ischemia, we hypothesized that antioxidant compounds might be able to regulate the expression of SUR1. Therefore, the effect of resveratrol (RSV) on SUR1 expression was evaluated in the BMEC cell line HBEC-5i subjected to oxygen and glucose deprivation (OGD) for 2 h followed by different recovery times. Different concentrations of RSV were administered. ROS production was detected with etidine, and protein levels were evaluated by Western blotting and immunofluorescence. Intracellular Na + levels and cellular swelling were detected by imaging; cellular metabolic activity and rupture of the cell membrane were detected by MTT and LDH release, respectively; and EMSA assays measured the activity of transcriptional factors. OGD/recovery increased ROS production induced the AKT kinase activity and the activation of SP1 and NFκB. SUR1 protein expression and intracellular Na + concentration in the HBEC-5i cells increased after a few hours of OGD. These effects correlated with cellular swelling and necrotic cell death, responses that the administration of RSV prevented. Our results indicate that the ROS/AKT/SP1-NFκB pathway is involved in SUR1 expression during OGD/recovery in BMEC of the blood-brain barrier. Thus, RSV prevented cellular edema formation through modulation of SUR1 expression.

The spontaneously hypertensive rat (SHR) is a model widely used to investigate the causal mechanisms of essential hypertension. The enhanced catecholamine (CA) release reported in adrenal glands from adult SHRs raised considerable interest for its possible implication in the genesis of hypertension. The use of powerful techniques such as calcium imaging, electrophysiology, and single-cell amperometry to monitor in real time the key steps in CA secretion has allowed a better understanding of the role of chromaffin cells (CC) in the pathophysiology of hypertension, although several questions remain. Additionally, the implementation of these techniques in preparations in situ, such as the acute adrenal gland slice, which maintains the microenvironment, cell-to-cell communication, and anatomical structure similar to that of the intact adrenal gland, yields data that may have even greater physiological relevance. Here, we describe the procedures to measure the blood pressure of rats in a noninvasive manner, how to obtain primary cultures of adrenal chromaffin cells and acute adrenal slices, and how to perform amperometric recordings and intracellular calcium imaging in these preparations.

Significance Statement The calcium-sensing receptor (CaSR) activates salt reabsorption through the sodium-chloride cotransporter (NCC) via the WNK4-SPAK pathway. Glucose and other sugars are positive allosteric modulators of the CaSR. This would be irrelevant for most cells that come into contact with glucose, except for the nephron segments beyond the proximal tubule. Using in vitro and in vivo models, this work shows that the arrival of glucose or fructose to the distal nephron modulates the CaSR due to a positive allosteric type II effect associated with activation of the WNK4-SPAK-NCC pathway. This study proposes a mechanism by which glucose or fructose delivered to the distal nephron contributes to renal salt retention by their allosteric effect on the CaSR. Background The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. Methods We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. Results HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. Conclusions Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

ABSTRACT Anti-N-methyl D-aspartic acid receptor (anti-NMDAR) encephalitis is caused by anti-NMDAR antibodies (Abs) that induce neurologic and psychiatric symptoms, explained mainly by NMDAR hypofunction. In the long-term, these Abs decrease surface NMDAR and NMDAR-mediated intracellular Ca 2+ ([Ca 2+ ]i) influx. However, there are contradictory findings regarding short-term mechanisms. We investigated NMDAR function in cultured neurons after 60 min treatment with three commercial, rabbit, anti-NMDAR Abs (anti-GluN1 extracellular (EC) domain; anti-GluN2B EC domain; and anti-GluN1 intracellular (IC) domain). The anti-GluN2B and anti-GluN1 IC Abs were previously reported to mimic patientś Ab effects in a rodent in vivo model and decreased NMDAR-mediated [Ca 2+ ]i entry after 24 h treatment in our cells. After 60 min incubation with anti-GluN2B or anti-GluN1 IC decreased the NMDAR-mediated [Ca 2+ ]i rise, whereas anti-GluN1 EC slightly increased it. Interestingly, all Abs induced p38 phosphorylation (p-p38). However, surprisingly, it was also elicited by a rabbit Ab directed against a non-NMDAR intracellular epitope, which also reduced NMDAR-mediated [Ca 2+ ]i entry. We further investigated the cellular mechanisms regulated by the anti-GluN2B Ab after 60 min. This Ab did not reduce surface NMDAR and p38 inhibition partially prevented its effect on NMDAR function. This Ab did not elicit per se an [Ca 2+ ]i rise, whereas NMDAR inhibitors 7DCK and MK-801 did not prevent p-p38. Nonetheless, 7DCK prevented NMDAR-mediated [Ca 2+ ]i reduction by the Ab, suggesting a role of GluN1 flux-independent signaling. These data indicate that anti-NMDAR and non-anti-NMDAR Ab modulate NMDAR function distinctly and p38 signaling in the short-term, and a role of a third-party mediator. Finally, our results suggest the involvement of NMDAR flux-independent signaling.

ALL is a highly aggressive subtype of leukemia that affects children and adults. Glucocorticoids (GCs) are a critical component of the chemotherapeutic strategy against T-ALL. Cases of resistance to GC therapy and recurrent disease require novel strategies to overcome them. The present study analyzed the effects of Dex, one of the main GCs used in ALL treatment, on two T-ALL cell lines: resistant Jurkat and unselected CCRF-CEM, representing a mixture of sensitive and resistant clones. In addition to nuclear targeting, we observed a massive accumulation of Dex in mitochondria. Dex-treated leukemic cells suffered metabolic reprogramming from glycolysis and glutaminolysis towards lipolysis and increased FAO, along with increased membrane polarization and ROS production. Dex provoked mitochondrial fragmentation and induced autophagy/mitophagy. Mitophagy preceded cell death in susceptible populations of CCRF-CEM cells while serving as a pro-survival mechanism in resistant Jurkat. Accordingly, preventing FAO or autophagy greatly increased the Dex cytotoxicity and overcame GC resistance. Dex acted synergistically with mitochondria-targeted drugs, curcumin, and cannabidiol. Collectively, our data suggest that GCs treatment should not be neglected even in apparently GC-resistant clinical cases. Co-administration of drugs targeting mitochondria, FAO, or autophagy can help to overcome GC resistance.

Hypertension is a multifactorial disease characterized by vascular and renal dysfunction, cardiovascular remodeling, inflammation, and fibrosis, all of which are associated with oxidative stress. We previously demonstrated cellular reactive oxygen species (ROS) imbalances may impact the structural and biochemical functions of blood cells and reported downregulation of β-dystroglycan (β-Dg) and overexpression of the epithelial sodium channel (ENaC) contribute to the pathophysiology of hypertension. In this study, we aimed to determine the expression of dystroglycans (Dg) and ENaC in platelet progenitors (megakaryocytes) and their surrounding niches. Thin sections of bone marrow from 5- and 28-week-old spontaneous hypertensive rats (SHR) were compared to age-matched normotensive rats (WKY). Cytometry and immunohistochemical assays demonstrated an oxidative environment in SHR bone marrow, characterized by high levels of myeloperoxidase and 3-nitrotyrosine and downregulation of peroxiredoxin II. In addition, transmission electron micrography and confocal microscopy revealed morphological changes in platelets and Mgks from SHR rats, including swollen mitochondria. Quantitative qRT-PCR assays confirmed downregulation of Dg mRNA and immunohistochemistry and western-blotting validated low expression of β-Dg, mainly in the phosphorylated form, in Mgks from 28-week-old SHR rats. Moreover, we observed a progressive increase in β-1 integrin expression in Mgks and extracellular matrix proteins in Mgk niches in SHR rats compared to WKY controls. These results indicate accumulation of ROS promotes oxidative stress within the bone marrow environment and detrimentally affects cellular homeostasis in hypertensive individuals.

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.

Intracellular Ca 2+ ([Ca 2+ ]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca 2+ -induced Ca 2+ -release (CICR) from internal stores, which boosts intracellular Ca 2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca 2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca 2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca 2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca 2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca 2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca 2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca 2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca 2+ signals of varying widths. Some even show early and late Ca 2+ components. Narrowing the Ca 2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca 2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca 2+ signal and the rate of exocytosis at the single-cell level.

Resting membrane potential is a bioelectric property of all cells. Multiple players govern this property, the ion channels being the most important. Ion channel dysfunction can affect cells' resting membrane potential and could be associated with numerous diseases. Therefore, the drug discovery focus on ion channels has increased yearly. In addition to patch-clamp, cell-based fluorescent assays have shown a rapid and reliable method for searching new ion channel modulators. Here, we used a cell-based membrane potential assay to search for new blockers of the Kv10.1, a potassium channel strongly associated with cancer progression and a promising target in anticancer therapy. We found that fluoxetine and miconazole can inhibit the Kv10.1 channel in the micromolar range. In contrast, BL-1249 potentiates Kv10.1 currents in a dose-dependent manner, becoming the first molecule described as an activator of the channel. These results demonstrate that cell-based membrane potential assay can accelerate the discovery of new Kv10.1 modulators.