Albumins as Extracellular Protein Nanoparticles Collaborate with Plasma Ions to Control Biological Osmotic Pressure

Abstract

Introduction: Plasma albumins as protein nanoparticles (PNs) exert essential functions in the control of biological osmotic pressure (OP), being involved in regulating water metabolism, cell morphology and cell tension. Understanding how plasma albumins and different electrolytes co-determine biological OP effects is crucial for correct interpretation of hemodynamic disorders, and practical treatment of hypo/hyper-proteinemia.

Methods: Optical measurement based on intermediate filament (IF) tension probe was used for real-time evaluation of transmembrane osmotic effects in live cells. Ion fluorescent probes were employed to evaluate intracellular ion levels, and a current clamp was used to measure membrane potential, thus exploring association of electrochemical and osmotic effects.

Results: Albumins are involved in regulation of intracellular osmolarity by a quantitative relationship. Extracellular PNs can alter membrane potentials by adsorbing counterions, induce production of intracellular PNs and further control the opening of ion channels and ion flow, contributing to electrochemical and osmotic re-equilibrium. Furthermore, various ions interplay with extracellular PNs, showing different osmotic effects: increased levels of calcium ions result in a hypotonic effect, whereas potassium ions induce hyper-osmolarity.

Conclusion: Extracellular PNs and Ca²⁺/K⁺ display antagonistic or synergetic effects in regulating biological OP. Live cells can spontaneously regulate osmotic effects through changing membrane potential and controlling intracellular ion content. Various plasma components need to be comprehensively analyzed, further developing a diagnostic index that considers the biological OP effects of various blood components and improves the evaluation of symptoms and diseases, such as calcium/potassium-hemodynamic disorders and edema.

Keywords: albumin; biological OP; electrochemical and osmotic re-equilibrium; intermediate filament tension; protein nanoparticle-ion interplay.

Graphical abstract

Figure 1

Relationships between extracellular OP gradient, GFAP tension, and intracellular OP. (A and B) A GFAP cpstFRET probe module was used to measure changes in IF tension in U87 cells. Representative images of normalized Cyan Fluorescence Protein (CFP)/FRET ratios of GFAP tension subjected to the NaCl solutions with different concentrations, including extracellular hypoosmolality (less than 300 mOsmol/kg) and hyperosmolality (higher than 300 mOsmol/kg) are shown. (C) Representative images of normalized CFP/FRET ratios of GFAP tension subjected to hyperosmotic mannitol solutions. Scale bar, 10 μm. (D) Linear correlation between GFAP tension and NaCl concentration. (E) Linear correlation between GFAP tension and mannitol concentration. (F) Cytoplasmic OP values as measured using a freezing point osmometer. (G) Intracellular PN count rates under different treatments. Data presented as mean ± SEM.

Figure 2

Quantitative analysis of IF tension regulated by albumin and divalent cations. (A) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic MHBSS solutions with different albumin concentrations (0, 10, 30, 50, 70 and 100 mg/mL). (B) Relationship between GFAP tension and albumin concentration, showing the parabolic correlation of the experimental concentrations and the linear correlation in the pathophysiologic ranges (10–100 mg/mL albumin). (C) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic solutions containing 50 mg/mL albumin (the normal physiological concentration) and different Ca²⁺ concentrations (mM as number indicators). (D) Relationship between GFAP tension and Ca²⁺ concentration in different experimental and pathophysiologic ranges. (E) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic MHBSS solutions containing 50 mg/mL albumin and different Mg²⁺ concentrations (mM as number indicators). (F) Relationship between GFAP tension and Mg²⁺ concentration in different ranges. Scale bar, 10 μm. Data presented as mean ± SEM.

Figure 3

Quantitative analysis of IF tension regulated by monovalent cations. (A) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic MHBSS solutions containing 50 mg/mL albumin and different K⁺ concentrations (mM as number indicators). (B) Linear relationship between GFAP tension and K⁺ concentration. (C) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic MHBSS solutions containing 50 mg/mL albumin and different Na⁺ concentrations (mM as number indicators). (D) Linear relationship between GFAP tension and Na⁺ concentration. Scale bar, 10 μm. Data presented as mean ± SEM.

Figure 4

Osmotic effects for live cells derived from changes in intracellular OP, PN count, membrane potential and intracellular ion levels. The cytoplasmic OP values in response to extracellular variations of albumin (A), 50 mg/mL albumin and Ca²⁺ (B), 50 mg/mL albumin and K⁺ (C) as determined using a freezing point osmometer. Intracellular PN counts under the effects of different extracellular components, including albumin (D), 50 mg/mL albumin and Ca²⁺ (E), 50 mg/mL albumin and K⁺ (F). Membrane potential was measured by the current-clamp method under treatments with albumin (G), 50 mg/mL albumin and Ca²⁺ (H), and 50 mg/mL albumin and K⁺ (I). Chloridion imaging presents the relative MQAE fluorescence intensity ratio of 15 min (F₁₅/F₀) under different concentrations of extracellular albumin (J), 50 mg/mL albumin and Ca²⁺ (K), 50 mg/mL albumin and K⁺ (L). An increase in intracellular Cl⁻ levels leads to a decrease in MQAE fluorescence value. Calcium imaging presents the relative FLUO-4 fluorescence intensity ratio of 15 min (F₁₅/F₀) for cells under different concentrations of extracellular albumin (M), 50 mg/mL albumin and Ca²⁺ (N), 50 mg/mL albumin and K⁺ (O). Data presented as mean ± SEM.

Figure 5

Albumin and Ca²⁺ co-regulate osmotic effects. (A) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic solutions with hyper/hypo-albumin and Ca²⁺. Scale bar, 10 μm. (B) Cytoplasmic OP values as measured using a freezing point osmometer. (C) Intracellular PN counts, ns, not significant. (D and E) Membrane potentials using the current-clamp method under different conditions. (F and G) Chloridion imaging micrographs and traces of relative MQAE fluorescence intensity ratio of 15 min (F₁₅/F₀). An increase in intracellular Cl⁻ level leads to a decrease of MQAE fluorescence value. (H and I) Calcium imaging micrographs and traces of relative FLUO-4 fluorescence intensity ratio of 15 min (F₁₅/F₀). Scale bar, 100 μm. Data presented as mean ± SEM.

Figure 6

Albumin and K⁺ synergistically regulate transmembrane osmotic effects. (A) Representative images and mean values of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic solutions with different albumin and K⁺ concentrations. Scale bar, 10 μm. (B) Cytoplasmic OP values. (C) Intracellular PN count, ns, not significant. (D and E) Membrane potentials using the current-clamp method under different conditions. (F and G) Chloridion imaging micrographs and traces of relative MQAE fluorescence intensity ratio of 15 min (F₁₅/F₀). (H and I) Calcium imaging micrographs and traces of relative FLUO-4 fluorescence intensity ratio of 15 min (F₁₅/F₀). Scale bar, 100 μm. Data presented as mean ± SEM.

Figure 7

Synergistical effects of albumin, Ca²⁺, and K⁺ on IF tension. Representative images (A) and mean values (B) of normalized CFP/FRET ratios of GFAP tension in cells subjected to isotonic solutions with different albumin, Ca²⁺, and K⁺ concentrations. Scale bar, 10 μm. Data presented as mean ± SEM.