Selective activation of intracellular β1AR using a spatially restricted antagonist
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ABSTRACT G-protein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of FDA-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of novel tools to separate GPCRs signaling at the plasma membrane from the ones initiated at intracellular compartments. We took advantage of the structural and pharmacological information available for β1-adrenergic receptor (β1AR), an exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell impermeable β1AR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited β1AR on the plasma membrane. In contrast, a cell permeable β1AR agonist containing a non-sulfonated fluorophore, efficiently inhibited both the plasma membrane and Golgi pools of β1ARs. Furthermore, the cell impermeable antagonist selectively inhibited the phosphorylation of downstream effectors of PKA proximal to the plasma membrane in adult cardiomyocytes while β1AR intracellular pool remained active. Our tools offer promising avenues for investigating compartmentalized β1AR signaling in various context, potentially advancing our understanding of β1AR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.Hydrogen peroxide (H2O2) is considered to be a mediator of apoptotic cell death but the mechanism by which it induces apoptosis is unclear. Here, we show that cells undergoing apoptosis from exposure to H2O2 display a significant decrease in intracellular concentration of superoxide (O2-) which is associated with a reduction of the intracellular milieu, as measured by an increase in the GSH/GSSG ratio and a decrease in intracellular pH. The notion that a decrease in intracellular O2- concentration triggers apoptosis is supported by the observation that H2O2-mediated apoptosis could be retarded in cells in which the intracellular O2- concentration is maintained at or above the cellular baseline level by inhibition of the major O2- scavenger superoxide dismutase (Cu/Zn SOD). Taken together, our observations indicate that a decrease in the intracellular O2- concentration, reduction and acidification of the intracellular milieu constitute a signal for H2O2-mediated apoptosis, thereby inducing a reductive as opposed to an oxidative stress.
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Intracellular Mechanical Drugs Today it is incontestable that cell mechanics is as important as cell biochemistry. However, although there is a plethora of biochemical drugs to study and treat cells, there is a lack of their mechanical counterparts. In article number 2109581, Teresa Suárez, José A. Plaza, and co-workers propose silicon chips as intracellular mechanical drugs to study how intracellular mechanical cues define cell function and fate with even future therapeutic potential applications.
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By means of light and electron microscopy, intracellular reparation has been studied after a local x-ray radiation of the rat paws (7.74 X 10(-1) Ci/kg) using radioprotectors (mexamin, cysteamin, ionol) and other chemical compounds (including membranoprotective ones). Restoration of the intracellular structures after x-ray burns proceeds more slowly and more complexly than reparation of the epidermis as a tissue system. To the slowly repairing intracellular formations belong mitochondria and, especially, internal mitochondrial membrane, as well as intercellular contacts. Under radiation mitochondria increase their volume at the expense of their three-fold swelling. Preliminary treatment of the skin with some of the compounds mentioned above decreases or completely prevents these changes. By means of the membranoactive chemical compounds, as well as by means of the known radioprotectors it is possible essentially to normalize the process of intracellular reparation and physiological regeneration of the ultrastructures, and in some cases, to stimulate reparative processes in them.
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Intracellular pH of the turtle bladder was measured with fluorescent probe 6-carboxyfluorescein (6-CF) diacetate. In isolated cells this probe provides reliable, reproducible and fast measurements of intracellular pH. The probe was mainly located in the cytosol and thus the values of intracellular pH mainly reflect cytosolic pH. The values of intracellular pH obtained with 6-CF were very similar to those measured with 14C-methylamine and 'null point' technique. The 6-CF technique was capable of detecting small changes in intracellular pH induced by acetazolamide. The intracellular pH of the mitochondrial-rich and granular cell fraction was not different. In addition to assessing intracellular pH of isolated cells, it was possible to monitor the intracellular pH of whole bladders continuously with 6-CF. Addition of CO2 to serosal solution decreased intracellular pH while perfusion with NH3 increased intracellular pH. Thus, 6-CF provides reliable and accurate measurements of intracellular pH in isolated cells and in whole bladders. This technique is capable of detecting rapid changes in intracellular pH and provides continuous monitoring of intracellular pH and thus should allow correlation of changes in urinary acidification with intracellular H+ concentration.
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1. The possible role of pH changes in mediating light adaptation in Limulus ventral photoreceptor cells was studied by intracellular injection of zwitterionic pH buffers. The intracellular concentration of buffer was estimated by inclusion of a radioactive marker in the injection solution. 2. The light‐induced increase of intracellular Ca2+ concentration was monitored by intracellular aequorin. The light‐induced increase of Ca2+ concentration was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 3. The progressive decrease in responsiveness during intracellular ionophoretic injection of Ca2+ was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 4. Photoreceptors of both Limulus and Balanus were impaled with two micropipettes and voltage clamped. Membrane current induced by a prolonged steady illumination declined from an early transient to a plateau. This delayed decline of current indicates a light‐induced reduction of sensitivity (i.e. light adaptation). The wave forms were similar before and after injection of pH buffer to an intracellular concentration of about 200 mM. 5. We conclude that it is unlikely that a light‐induced change of cytosolic pH mediates light adaptation in Limulus (and Balanus) photoreceptors.
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