title: Opposite effects of pressurized steady versus pulsatile perfusion on vascular endothelial cell cytosolic pH: role of tyrosine kinase and mitogen-activated protein kinase signaling.
abstract: Endothelial cytosolic pH (pH(i)) modulates ion channel function, vascular tone, and cell proliferation. Steady shear induces rapid acidification in bicarbonate buffer. However, in vivo shear is typically pulsatile, potentially altering this response. We tested effects and mechanisms of pH(i) modulation by flow pulsatility, comparing pressurized steady versus pulse-flow responses in bovine aortic endothelial cells cultured within glass capillary tubes. Cells were loaded with the fluorescent pH(i) indicator carboxy seminaphthorhodafluor-1 and perfused with physiological pulsatile pressure and flow generated by a custom servo-control system. Raising mean pressure from 0 to 90 mm Hg at 0.5 mL/min steady flow in bicarbonate buffer induced sustained acidification (-0.33+/-0.09 pH units, P<0.01). A subsequent increase in steady flow resulted in further acidification. In contrast, if mean pressure and flow were unchanged but perfusion made pulsatile, pH(i) rose +0.3+/-0.03 (P<0. 0001) over 30 to 60 minutes. HCO(3)(-) removal and use of acid/base exchange inhibitors 5-(N-ethyl-N-isopropyl)amiloride or diisothiocyanato stilbene disulfonic acid identified both extracellular Na(+)-independent Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers as activated by static pressure, whereas pulsatility activated extracellular Na(+)-dependent Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers to raise pH(i). Pulse-perfusion alkalinization occurred with or without flow reversal and increased 1.6-fold in Ca(2+)-free buffer. Inhibition of c-Src tyrosine kinase (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo [3,4-d]pyrimidine; PP2) or MEK-1 (mitogen-activated protein kinase [MAP]/extracellular signal-regulated kinase [ERK]-1) (PD98059, blocking ERK1/2) blocked or reversed the pulsatile-flow pH(i) change to acidification. In contrast, PP2 had no effect on steady flow acidification, whereas MEK-1 inhibition converted it to alkalinization. Thus, pulsatile and steady flow trigger opposite effects on endothelial pH(i) by differential activation of acid/base exchangers linked to c-Src and MAP kinase phosphorylation, but not to Ca(2+). These data highlight specific signaling responses triggered by phasic shear profiles.
authors.affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, MD, USA.
authors.forename: I S
authors.initials: IS
authors.lastname: Wittstein
authors.forename: W
authors.initials: W
authors.lastname: Qiu
authors.forename: R C
authors.initials: RC
authors.lastname: Ziegelstein
authors.forename: Q
authors.initials: Q
authors.lastname: Hu
authors.forename: D A
authors.initials: DA
authors.lastname: Kass
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006706:Homeostasis; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D010477:Perfusion / Q000379:methods*; D011312:Pressure; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D015398:Signal Transduction; D019061:src-Family Kinases / Q000502:physiology
chemical_list: D006859:Hydrogen; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium