In this second of three papers, we examine the morphometry red blood cells (RBCs) from our laboratory mouse strain (C57BL/6Case) and RBC-membrane proteomics. paper #1, using stopped-flow absorbance-spectroscopy to ascertain rate constant for oxyhemoglobin (HbO 2 ) deoxygenation (k HbO2 ), find substantial reductions in k with (1) membrane-protein inhibitors p-chloromercuribenzenesulfonate (pCMBS) or 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS); (2) knockouts aquaporin-1 (AQP1-KO),...

Many have believed that oxygen (O 2 ) crosses red blood cell (RBC) membranes by dissolving in lipids offer a finite resistance to diffusion or, alternatively, no at all. However, analyses of hemoglobin (Hb) absorbance spectra during O off-loading from mouse RBCs indicate RBC do indeed diffusion, and the would be far higher if not for presence -permeable channels. Two agents, both excluded interior, markedly slow off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces membrane...

Abstract In this third of three papers, we develop a reaction-diffusion model for O 2 offloading from red blood cell (RBC), treated as sphere with diameter approximating RBC thickness. Stopped-flow (SF) analysis ( paper #1 ) hemoglobin/oxyhemoglobin (Hb/HbO absorbance spectra during efflux intact murine RBCs show that membrane-impermeant inhibitor p-chloromercuribenzenesulfonate (pCMBS) reduces the HbO -deoxygenation rate constant k by ∼64%. SF experiments shows falls (i) 9% aquaporin-1...

Aquaporin-4 (AQP4) plays an important role in regulating water exchange across the blood-brain barrier (BBB) and brain-cerebrospinal fluid interface. Studies on AQP-4 knockout mice (AQP4-KO) have reported considerable protection from brain edema induced by acute intoxication ischemic stroke, identifying AQP4 as a potential target for therapeutic interventions. However, long-term effects of chronic suppression are yet to be elucidated. In current study, we evaluated physiological structural...

Fungiform papillae are formed as patterned rows on the surface of anterior tongue at early organogenesis and contain one taste bud in each papilla to form important sensory organs. Despite essential role Wnt/β-catenin signaling controlling development fungiform papillae, universal function Wnt ligands initiation placode has not been completely elucidated. Here, by Shh (Cre) -mediated oral epithelial deletion Wntless (Gpr177), a regulator for intracellular trafficking, we demonstrate that an...

We report a novel carbonic-anhydrase (CA) assay and its use for quantitating red-blood-cell (RBC) lysis during stopped-flow (SF) experiments. combine two saline solutions, one containing HEPES/pH 7.03 the other, ~1% CO2/44 mM HCO3−/pH 8.41, to generate an out-of-equilibrium CO2/HCO3− solution ~0.5% CO2/22 ~7.25 (10C) in SF reaction cell. CA catalyzes relaxation of extracellular pH ~7.50: HCO3− + H+  CO2 H2O. Proof-of-concept studies (no intact RBCs) show that pH-relaxation rate constant...

Several aspects of the cell biology cystic fibrosis (CF) epithelial cells are altered including impaired lipid regulation, disrupted intracellular transport, and microtubule regulation. It is unclear how loss transmembrane conductance regulator (CFTR) function leads to these differences. hypothesized that CFTR regulation carbonic anhydrase (CA) activity resulting in cellular phenotypic changes. In this study, it demonstrated CA2 protein expression reduced CF model cells, primary mouse nasal...

Abstract Many have believed that oxygen (O 2 ) crosses red blood cell (RBC) membranes by dissolving in lipids offer no resistance to diffusion. However, using stopped-flow (SF) analyses of hemoglobin (Hb) absorbance spectra during O off-loading from mouse RBCs, we now report most traverses membrane-protein channels. Two agents excluded the RBC interior markedly slow off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces inferred membrane permeability ( P Membrane ∼82%, and...

It is well known that there are at least three families of membrane proteins—aquaporins (AQPs), rhesus (Rh) proteins, and urea transporters (UTs)—function as channels for CO 2 or NH 3 . In human red blood cells (RBCs), AQP1 the Rh complex (mainly RhAG) together account ~90% permeability. We hypothesize most O crosses RBC membranes through polytopic proteins also act channels. quantitated efflux mouse using stopped‐flow (SF) absorbance spectroscopy to determine saturation hemoglobin (Hb). For...

Lipid metabolism plays an important role in the occurrence and development of tumor cells. The normal cells provide energy through lipids circulation system, while their own de novo synthesis. This abnormal lipid not only supplies for to rapidly proliferate, but also related are involved molecular signal pathways tumor. Increasing studies have found that there is a close relationship between Some key enzymes process metabolism, such as fatty acid synthase (FASN) stearoyl-coenzyme A...

The established role of NBCe1‐A (SLC4A4) is to cotransport Na + and HCO 3 − (or more likely, CO = ). We present evidence that also conducts 2 . record the ionic current carried by ( I NBC )—a direct measure activity, while simultaneously assessing traffic through from maximal rates intracellular pH (pH i ) changes, (dpH /dt) max employ a continuous‐flow, rapid‐mixing approach for generating out‐of‐equilibrium (OOE) /HCO solutions control [CO ], [HCO extracellular o individually in bath....

We are investigating the permeability of red blood cell (RBC) plasma membrane to various gases. Our laboratory was first establish that water channel aquaporin1 (AQP1) can also mediate CO 2 transport. Other research groups later showed AQPs conduct NH 3 and rhesus (Rh) proteins . Collectively AQP1 RhAG account for ~90% human RBCs. In other work presented at this meeting we molecular basis O transport (“Oxygen Channels in Red Blood Cells”, Pan Zhao et al. ). That study employs from wild‐type...

O 2 diffusion across red blood cell (RBC) membranes is a critically important physiological process for the maintenance of life. Our previous work on murine RBCs ( bioRxiv. doi: 10.1101/2020.08.28.265066 ) showed that (a) genetic deletion aquaporin-1 (AQP1) and Rh complex (mainly RhAG) together reduce permeability RBC P M, O2 by ~55%, (b) double knockout (dKO) AQP1 RhAG plus pCMBS (sulfhydryl reagent nonspecific inhibitor membrane proteins nonetheless excluded from interior) reduces ~91%. In...

The dogma that all gases move through red blood cell (RBC) membranes by dissolving in and diffusing the membrane lipid is no longer correct. Our preliminary data on mouse RBCs genetically deficient both aquaporin‐1 (AQP1) Rh complex (mainly RhAG) suggest these two proteins account for ~60% of O 2 permeability. Using stopped‐flow (SF) analysis hemoglobin (Hb) absorbance spectra, we now examine effect aging rate constant Hb deoxygenation ( k HbO2 ) from WT mice AQP1 RhAG (double knockout,...

We now recognize that the paradigm all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through lipid phase is not correct. Work on human RBCs genetically deficient aquaporin‐1 (AQP1) or Rh complex (mainly RhAG) showed these two proteins account for ~90% of membrane CO 2 permeability ( P M,CO2 ), 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces . used stopped‐flow (SF) analysis hemoglobin (Hb) absorbance spectra as we mixed oxygenated with an...

The traditional view had been that all O 2 moves through red blood cell (RBC) membranes by dissolving in and diffusing the membrane lipid. In a previous abstract, we reported data obtained using stopped‐flow analysis of hemoglobin (Hb) absorbance spectra on RBCs from wild‐type (WT) vs. double‐knockout (dKO) mice genetically deficient both aquaporin‐1 (AQP1) rhesus (Rh) protein RhAG. We found rate constant Hb deoxygenation ( k HbO2 )—which includes intra/extracellular diffusion...

The diffusion of O2 across red blood cell (RBC) membranes is critically important for the delivery to peripheral tissues. Although traditional view had been that all moves through RBC by dissolving in and diffusing membrane lipid, our recent work on murine RBCs indicates most traversing via protein channels (bioRxiv. doi:10.1101/2020.08.28.265066). In stopped-flow (SF) kinetics experiments, we use absorbance spectrum hemoglobin (Hb) monitor rate constant offloading from Hb (kHbO2). mice...

The diffusion of O2 across the red blood cell (RBC) membrane is critical for life. Although traditional view had been that all moves through RBC membranes by dissolving in and diffusing lipid, our recent work (bioRxiv. doi:10.1101/2020.08.28.265066) indicates this not case. Using stopped-flow absorbance spectroscopy to monitor hemoglobin (Hb) spectrum intact RBCs, we found that, compared with RBCs from wild-type (WT) mice, those double knockout (dKO) mice genetically deficient both...