The mechanism that promotes transition from the ATP- to the ADP-liganded state of ATP-sensitive K+ (KATP) channels and consequent channel opening in a cytosolic environment of high ATP concentration has yet to be understood. A mechanism examined here that could reverse the ATP-inhibited state is based on the action of adenylate kinase to catalyze phosphoryl transfer between ATP and AMP, resulting in transformation of ATP into ADP. In membrane patches excised from guinea pig cardiomyocytes, AMP alone did not affect channel behavior but increased the open probability of ATP-inhibited KATP channels. This required MgCl2 and a hydrolyzable form of ATP and was prevented by P1,P5-di-adenosine-5'-pentaphosphate, an inhibitor of adenylate kinase. The single channel amplitude and kinetics of channel openings induced by the ADP-generating substrates of adenylate kinase, AMP and MgATP, were indistinguishable from the biophysical properties of the KATP channel exhibited after addition of MgADP. In whole cell voltage-clamped cardiomyocytes, introduction of exogenous adenylate kinase along with millimolar MgATP and AMP induced a K+ current that was suppressed by a sulfonylurea blocker of KATP channels. Enriched sarcolemmal membrane preparations were found to possess ATP.AMP phosphotransferase activity with properties attributable to an extramitochondrial isoform of adenylate kinase. These results indicate that adenylate kinase is a naturally occurring component of sarcolemmal membranes that could provide dynamic governance of KATP channel opening through its phosphoryl transfer catalytic action in the microenvironment of the channel.

Las enfermedades cardiovasculares: un problema de salud pública y un reto global

Diarrhea induced by Escherichia coli heat-stable enterotoxin (ST_a) is mediated by a receptor guanylyl cyclase cascade. The present study establishes that an intracellular nucleotide-dependent pathway disrupts toxin-induced cyclic GMP (cGMP) production and the associated chloride (Cl⁻) flux that underlie intestinal secretion. Incubation of Caco 2 human intestinal epithelial cells with the nucleoside analog 2-chloroadenosine (2ClAdo) resulted in a concentration- and time-dependent inhibition of toxin-induced cGMP production. Inhibition of cGMP production correlated with the metabolic conversion of 2ClAdo to 2-chloroadenosine triphosphate. The effect of 2ClAdo did not reflect activation of adenosine receptors, inhibition of adenosine deaminase, or modification of the binding or distribution of ST_a receptors. Guanylyl cyclase activity in membranes prepared from 2ClAdo-treated cells was inhibited, in contrast to membranes from cells not exposed to 2ClAdo, demonstrating that inhibition of guanylyl cyclase C (GCC) was mediated by a noncompetitive mechanism. Treatment of Caco 2 cells with 2ClAdo also prevented ST_a-induced Cl⁻ current. Application of 8-bromo-cGMP, the cell-permeant analog of cGMP, to 2ClAdo-treated cells reconstituted the Cl⁻ current, demonstrating that inhibition of Cl⁻ flux reflected selective disruption of ligand stimulation of GCC rather than the chloride channel itself. Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing the utility of this pathway to elucidate the mechanisms regulating ST-dependent guanylyl cyclase signaling and intestinal fluid homeostasis. In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for enterotoxigenic diarrhea.

Cardiovascular diseases and cancer are leading global causes of morbidity and mortality, necessitating advances in diagnosis and treatment. Doxorubicin (Doxo), a potent chemotherapy drug, causes long-term heart damage due to cardiotoxicity. Small extracellular vesicles (sEVs) carry bioactive molecules—such as proteins, lipids, and nucleic acids—that can modulate gene expression and signaling pathways in recipient cells, including cardiomyocytes. Through the delivery of cytokines, microRNAs, and growth factors, sEVs can influence cell survival, which plays a critical role in the development of cardiotoxicity. This study investigates the role of sEVs derived from breast cancer cells treated or not with Doxo and their potential to induce cardiomyocyte damage, thereby contributing to cardiotoxicity. We isolated sEVs from MCF-7 cells treated or not to Doxo using ultracentrifugation and characterized them through Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), and Western Blotting (WB) for the markers CD63, CD81, and TSG101. We analyzed cytokine profiles using a Multiplex Assay and Cytokine Membrane Array. We exposed Guinea pig cardiomyocytes to different concentrations of sEVs. We assessed their viability (MTT assay), shortening, reactive oxygen species (ROS–DHE dye) production, mitochondrial membrane potential (JC-1 dye), and calcium dynamics (FLUO-4 dye). We performed statistical analyses, including t-tests, ANOVA, Cohen’s d, and η² to validate the robustness of the results. Treatment of MCF-7 cells with 0.01 μM Doxorubicin resulted in increased sEVs production, particularly after 48 h of exposure (~1.79 × 10⁸ ± 2.77 × 10⁷ vs. ~5.1 × 10⁷ ± 1.28 × 10⁷ particles/mL, n = 3, p = 0.0019). These sEVs exhibited protein profiles in the 130–25 kDa range and 93–123 nm sizes. They carried cytokines including TNF-α, IL-1β, IL-4, IFN-γ, and IL-10. Exposure of cardiomyocytes to sEVs (0.025 μg/mL to 2.5 μg/mL) from both Doxo-treated and untreated cells significantly reduced cardiomyocyte viability, shortened cell length by up to 20%, increased ROS production, and disrupted calcium homeostasis and mitochondrial membrane potential, indicating severe cellular stress and cardiotoxicity. These findings suggest that Doxo enhances sEVs production from breast cancer cells, which plays a key role in cardiotoxicity through their cytokine cargo. The study highlights the potential of these sEVs as biomarkers for early cardiotoxicity detection and as therapeutic targets to mitigate cardiovascular risks in chemotherapy patients. Future research should focus on understanding the mechanisms by which Doxorubicin-induced sEVs contribute to cardiotoxicity and exploring their diagnostic and therapeutic potential to improve patient safety and outcomes in cancer therapy.