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(Circulation Research. 2000;86:1230.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Opposite Effects of Pressurized Steady Versus Pulsatile Perfusion on Vascular Endothelial Cell Cytosolic pH

Role of Tyrosine Kinase and Mitogen-Activated Protein Kinase Signaling

Ilan S. Wittstein¹, Weiping Qiu¹, Roy C. Ziegelstein, Qinghua Hu, David A. Kass

From the Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Md.

Correspondence to David A. Kass, MD, Halsted 500, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail dkass{at}bme.jhu.edu

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. HCO₃^- 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^--HCO₃^- and Na⁺-H⁺ exchangers as activated by static pressure, whereas pulsatility activated extracellular Na⁺–dependent Cl^--HCO₃^- and Na⁺-H⁺ exchangers to raise pH_i. Pulse-perfusion alkalinization occurred with or without flow reversal and increased 1.6-fold in Ca²⁺-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 Ca²⁺. These data highlight specific signaling responses triggered by phasic shear profiles.

Key Words: endothelium • acid-base • pulsatile shear • mitogen-activated protein kinase • tyrosine kinase

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