Howell, N. | Search bioRxiv

bioRxiv2025

Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

BackgroundAngiotensin type 2 receptor (AT2R) activation promotes natriuresis in renal proximal tubule cells (RPTCs) counteracting sodium retention stimulated by the AT1R. Early signaling events mediating the natriuretic response with AT2R activation in RPTCs are currently unknown. Our previous research suggested protein phosphatase 2A (PP2A) functions downstream of the AT2R. In this study, we investigated the interaction of PP2A regulatory subunit B55 with the AT2R and requirement for B55 in AT2R signaling and natriuresis. Methods and ResultsRats were subjected to renal interstitial (RI) infusion of vehicle or Compound 21 (C21), a non-peptide specific AT2R agonist, and kidney sections were probed for interactions between PP2A subunits and the AT2R using a proximity ligation assay. A dramatic 6-fold increase in AT2R-B55 interaction in apical brush border membranes of RPTCs was observed with C21 stimulation. In vitro binding of purified AT2R and B55 supported a direct interaction between these two proteins. To determine the requirement for B55 in renal AT2R signaling, siRNA targeting B55 was administered to rats in vivo by RI infusion which resulted in a [~]70% decrease in B55 in RPTCs but not distal tubules. In rats with B55 knockdown in RPTCs, natriuresis in response to C21 was abolished. Simultaneously, C21-elicited AT2R redistribution to and sodium transporter Na+/H+ exchanger-3 (NHE3) retrieval from apical brush border membranes was lost, as observed with confocal immunofluorescence microscopy. Consistent with impaired AT2R signaling, B55 knockdown prevented c-Src phosphorylation in response to C21. B55 knockdown also led to a dramatic 4 to 6-fold increase in AT2R co-localization with the lysosomal marker LAMP1, and a 50% reduction in AT2R co-localization with EEA1 and Rab 7, markers for early and late endosomes, respectively. ConclusionsPP2A B55 directly binds to the activated AT2R and is required for AT2R-elicited natriuresis, AT2R signaling and intracellular trafficking in RPTCs.

physiology↗

bioRxiv2023

Neutron capture enhances dose and reduces cancer cell viability in and out of beam during helium and carbon ion therapy.

PurposeNeutron Capture Enhanced Particle Therapy (NCEPT) is a proposed augmentation of charged particle therapy which exploits thermal neutrons generated internally, within the treatment volume via nuclear fragmentation, to deliver a biochemically targeted radiation dose to cancer cells. This work is the first experimental demonstration of NCEPT, performed using both carbon and helium ion beams with two different targeted neutron capture agents (NCAs). Materials and MethodsHuman glioblastoma cells (T98G) were irradiated by carbon and helium ion beams in the presence of NCAs, [10B]-BPA and [157Gd]-DOTA-TPP. Cells were positioned within a PMMA phantom either laterally adjacent to, or within, a 100x100x60 mm spread out Bragg peak (SOBP). The impact of NCAs and location relative to the SOBP on the cells was measured by cell growth and survival assays in six independent experiments. Neutron fluence within the phantom was characterised by quantifying the neutron activation of gold foil. ResultsCells placed inside the treatment volume reached 10% survival by 2 Gy of C or 2-3 Gy of He in the presence of NCAs compared to 5 Gy of C and 7 Gy of He with no NCA. Cells placed adjacent to the treatment volume showed a dose-dependent decrease in cell growth when treated with NCAs, reaching 10% survival by 6 Gy of C or He (to the treatment volume), compared to a no detectable effect on cells without NCA. The mean thermal neutron fluence at the centre of the SOBP was approximately 2.2x109 n/cm2/Gy(RBE) for the carbon beam and 5.8x109 n/cm2/Gy(RBE) for the helium beam and gradually decreased in all directions. ConclusionsThe addition of NCAs to cancer cells during C and He beam irradiation has a measurable impact on cell survival and growth in-vitro. Through the capture of internally generated neutrons, NCEPT introduces the concept of a biochemically targeted radiation dose to charged particle therapy. NCEPT enables the established pharmaceuticals and concepts of neutron capture therapy to be applied to a wider range of deeply situated and diffuse tumours, by targeting this dose to micro-infiltrates and cells outside of defined treatment regions. These results also demonstrate the potential for NCEPT to provide an increased dose to tumour tissue within the treatment volume, with a reduction in radiation doses to off target tissue.

biophysics↗