Sodium and cell rigidity Hans Oberleithner Institute of Physiology II University Münster, Germany Calcium and the cytoskeleton, Minisymposium, Casapueblo, November 3, 2007; 11:30 a.m.
This is what we need This is what we eat
20,000 Deaths from stroke in Montevideo per year Uruguay Meneton et al; Physiol Rev 2005
Hypertension 2005; 45: 98-102
3g 6g 9g
Some physiology The mineralocorticoid hormone aldosterone controls salt transport in the kidney retains salt in the extracellular compartments and thus maintains arterial blood pressure.
Some more physiology Besides kidney, the cardiovascular system is a target for aldosterone Myocardium Vascular smooth muscle endothelium
Endothelium (Felix) imaged in fluid Image size: 180 x 180 µm Image resolution: 1024 x 1024 lines Time to obtain the image: 30 min Instrument: BioScope II from Veeco
Epithelial sodium channels (ENaC) are found in endothelia and they are upregulated by aldosterone = Na + = Ca 2+ amiloride binding ouside Ca 2+ cannot enter inside
1,250 ENaC found in the apical surface of an endothelial cell No aldosterone Kusche et al Pfügers Arch 2007 10 µm
1,750 ENaC found in the apical surface of an endothelial cell aldosterone +29% Kusche et al Pfügers Arch 2007 10 µm
Is salt an endothelial stiffener and is ENaC involved?
Force distance curves performed in a single endothelial cell
For details go to: www.nanophysiology.de
Endothelial cell stiffness? 120 130 140 150 160 170 Extracellular sodium concentration (mm) low sodium normal sodium high sodium
Three force distance curves
laser 500 3. deform cantilever AFM tip Cantilever deflection (nm) Δ 50 nm Δ 54 nm Δ 78 nm 200 nm 200 nm 200 nm 2. touch 1. approach 145 mm Na + 135 mm Na + 125 mm Na + 0-400 Cell membrane indentation depth Distance between AFM tip and cell surface 0 500 nm
Change in endothelial cell stiffness (%) 60 50 40 30 20 10 0-10 120 130 140 150 160 170 Oberleithner et al PNAS 2007 Eplerenone treated cells Aldosterone treated cells vertical force Extracellular sodium concentration (mm)
A rise in extracellular sodium transiently increases cell volume and the ENaC blocker amiloride prevents this response
A B Single cell line scan versus time h line scan height profile h 200 nm 1 min 135 Na + 150 mm Na + (constant osmolarity) D Transient increase in cell height (nm) Transient increase in cell height (nm) 300 250 200 150 100 50 0 P = 0.001 135 to 150 mm Na + 135 to 150 mm Na + + amiloride C Single cell line scan versus time height profile h < 50 nm 135 Na + 150 mm Na + + amiloride (const. osmolarity)
Amiloride prevents sodium-mediated cell stiffening
Change in endothelial cell stiffness (%) 70 60 50 40 30 20 10 0 Human endothelium, pretreated with aldosterone Amiloride resistent Amil Amiloride sensitive Amil 135 mm sodium 150 mm sodium
High sodium reduces endothelial deformability
Force Scan
pretreated with aldosterone 2 µm 2 µm 135 mm sodium 150 mm sodium 0 20 40 60 80 µm 0 soft stiff 20 40 60 80 µm 2 µm 2 µm 25 µm A constant shear force of 5 nn is applied!
Is a stiff endothelial cell functionally inferior to a soft one?
Blood flow Deformation NO Vascular smooth muscle relaxes
Blood flow Deformability NO Blood vessel does not relax
Aortic endothelial cells GM 7373 (more NO metabolism) cultured in flasks little culture medium low/high sodium +/- aldosterone reduced in proteins (improves NO analysis) flasks tilted rhythmically for 24h (creates shear forces) medium filtered lyophilized resuspended nitrite measurement/griess reagent spectrophotometry
Nitrite concentration in the culture medium (µm) 6 5 4 3 2 1 0 135 mm Na + 135 mm Na + 150 mm Na + 150 mm Na + + aldosterone + aldosterone * Oberleithner et al PNAS 2007
plasma sodium concentration increases beyond 140 mm in physiological conditions, i.e. that aldosterone is available sodium volume pulsatile force endothelial cell nitric oxide deformability vascular smooth muscle cell tension increases Oberleithner et al PNAS 2007
Δ sodium Δ volume Δ stiffness Δ NO Δ blood pressure
Δ sodium Δ volume Δ stiffness Δ NO Δ blood pressure
Hypertension 2005; 45: 98-102
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Where the hell is Münster? Germany
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Münster Christoph Riethmüller...NO Hermann Schillers...AFM Martin Hausberg...Hypertension Kristina Kusche-Vihrog...ENaC Graham MacGregor... Idea about sodium Hugh de Wardener... kept us going