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© 1996 American Heart Association, Inc.
Articles |
NO Flow Helps Clear Murky Waters?
From the Department of Pathology, University of Chicago School of Medicine, Chicago, Ill.
Correspondence to Dr Peter Davies, Department of Pathology, University of Chicago School of Medicine, 5841 S Maryland Ave, Chicago, IL 60637.
Key Words: Editorials • nitric oxide • calcium • shear stress • tyrosine kinase • endothelium
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Introduction |
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 Top Introduction References |
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The relationships between blood flow and arterial tone have fascinated physiologists for decades. Increased and decreased flows result in vessel relaxation and contraction, respectively. Consequently, when pharmacological or neurochemical events constrict an artery, the increased flow and shear stress stimulate a compensatory vasorelaxation to restore the vessel tone, returning the average shear stress to near its previous level. The endothelium is now recognized as the principal vascular signal transducer that responds to changes of flow,1 including the release of nitric oxide (NO). Endothelium-derived NO then acts on smooth muscle guanylate cyclase to relax the vessel. There is much evidence to suggest that shear stress is the principal stimulus for activation of endothelial NO synthase (eNOS), the enzyme responsible for converting arginine to citrulline and NO. However, the mechanisms that lead from hemodynamic forces at the endothelial luminal surface to the activation of eNOS remain murky. Although sustained increases in flow stimulate eNOS gene expression, these transcriptional mechanisms are likely to be downstream from the rapid (and reversible) signaling required for minute-to-minute adjustments of arterial diameter. Efforts have therefore been concentrated on second messenger pathways studied principally in cultured cells. In this issue of Circulation Research, Ayajiki et al2 (in Rudi Busse's group at Frankfurt, Germany) report investigations of flow-induced NO release in arterial tissues. They confirm earlier signaling mechanisms elucidated in vitro while raising a number of questions pertinent to real tissues.
In cultured cells, NO production in response to shear stress is
biphasic.3 The initial peak of NO release is
Ca2+ dependent but independent of shear stress magnitude;
in contrast, the sustained phase of NO release is Ca2+
independent and dependent on shear stress magnitude.3 4 5
Different mechanisms of eNOS stimulation by flow and by agonists are
suggested by the finding that agonist-mediated increases are
Ca2+ dependent.6 Shear stress appears to
stimulate Ca2+-dependent NO release in periodic bursts at
15-minute intervals,4 whereas the
Ca2+-independent NO release is continuous and sustained.
Ayajiki et al2 extended such investigations to a bioassay
system in which shear stress was increased by inducing vasoconstriction
in an endothelium-intact donor segment of rabbit
iliac artery while maintaining a constant luminal perfusion rate. At
first they found biphasic Ca2+-dependent and
Ca2+-independent responses to shear stress–induced NO
release in the arterial tissues, consistent with
the in vitro data. However, when the arterial segments were
restored to their in vivo lengths, the initial
Ca2+-dependent phase was abolished without effect on the
Ca2+-independent sustained phase of NO release. They
suggest that shear stress–induced NO production proceeds
principally through a Ca2+-independent mechanism in intact
arteries. The differences may be related to overt or subtle changes in
endothelial cell morphology, perhaps related to
cytoskeletal organization. Recently, Barbee et al7 have
demonstrated large differences in the gradients of shear stress that
were related to the surface topography of individual
endothelial cells and that may be related to
heterogeneous cellular responses to flow.8 In
the study of Ayajiki et al,2 restoration of the
arterial length may change the morphology of the
endothelial cells below a critical threshold for
Ca2+-dependent, but not Ca2+-independent, NO
release.
The second set of interesting results from Ayajiki et al2 concerns the mechanotransduction pathways related to NO release. Last year in Circulation Research, Tseng et al5 (in Brad Berk's group at Seattle, Wash) demonstrated in cultured endothelium that shear stress stimulated the phosphorylation of 42- and 44-kD mitogen-activated protein kinases (MAP kinases) within 5 minutes. MAP kinase activation was independent of Ca2+ and was preventable by nonhydrolyzable GDP analogues (suggesting an upstream G-protein requirement) and by inhibition or downregulation of protein kinase C. In their present study, Ayajiki et al2 show that the tyrosine kinase inhibitor erbstatin A (herbimycin A) completely abolished shear stress–dependent NO production in arteries, suggesting an important role for tyrosine phosphorylation. Parallel studies in cultured endothelial cells confirmed not only the phosphorylation of MAP kinases after exposure to shear stress but also demonstrated enhanced tyrosine phosphorylation of a group of cytoskeletal proteins; phosphorylation of all these proteins was inhibited by erbstatin. In short, these and previous data suggest that tyrosine phosphorylation is required for the activation of eNOS. It should be noted, however, that eNOS (in common with other NO synthases) contains consensus sequences for phosphorylation by protein kinases A and C and calmodulin kinase II,9 and it is still unclear whether a G protein–coupled MAP kinase pathway is specifically required for the regulation of eNOS.
So the waters remain rather muddy. Perhaps serpentine (G
protein–linked) receptors are connected to the MAP kinase pathways
through 
and ß
subunits of G proteins, as occurs for the
transmission of growth and differentiation signals from the cell
surface to the nucleus.10 Perhaps Ras, a
GTPase, is a key molecular switch between the surface signal and the
MAP kinases11 ; the small G proteins rac and
rho linked to integrins could play some role, and perhaps
Raf is also involved.12 Ayajiki et
al2 referred to unpublished observations that shear stress
markedly alters the physical characteristics of the particulate eNOS,
enhancing its membrane binding. eNOS contains a myristoylation site
near the NH2 terminal that may anchor the enzyme to the
plasma membrane.9 It remains to be seen whether eNOS
activation through membrane interactions is related to the signaling
and phosphorylation responses discussed above.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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1. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev.. 1995;75:519-560.
2.
Ayajiki K, Hindermann M, Hecker M, Fleming I, Busse
R. Intracellular pH and tyrosine phosphorylation
but not calcium determine shear stress–induced nitric oxide
production in native endothelial cells.
Circ Res.. 1996;78:750-758.
3. Kuchan MJ, Frangos JA. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am J Physiol. 1994;266(Cell Physiol 35):C628-C636.
4.
Kanai AJ, Strauss HC, Truskey GA, Crews AL, Grunfeld
S, Malinski T. Shear stress induces ATP-independent transient
nitric oxide release from vascular endothelial cells,
measured directly with a porphyrinic microsensor. Circ
Res.. 1995;77:284-293.
5.
Tseng H, Peterson TE, Berk BC. Fluid shear
stress stimulates mitogen-activated protein kinase in
endothelial cells. Circ Res.. 1995;77:869-878.
6. MacArthur H, Hecker M, Busse R, Vane JR. Selective inhibition of agonist-induced but not shear stress–dependent release of endothelial autacoids by thapsigargin. Br J Pharmacol.. 1993;108:100-105. [Medline] [Order article via Infotrieve]
7.
Barbee KA, Mundel T, Lal R, Davies PF.
Subcellular distribution of shear stress at the surface of
flow-aligned and nonaligned endothelial
monolayers. Am J Physiol. 1995;268:H1765-H1772.
8. Davies PF, Mundel T, Barbee KA. A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro. J Biomech. 1995;28:1553-1560. [Medline] [Order article via Infotrieve]
9. Sessa WC. The nitric oxide synthase family of proteins. J Vasc Res.. 1994;31:131-143. [Medline] [Order article via Infotrieve]
10.
Crespo P, Xu N, Simons WF, Gutkind JS.
Ras-dependent activation of MAP kinase pathway mediated by G
protein ß subunits. Nature. 1994;369:418-420. [Medline]
[Order article via Infotrieve]
11.
Hall A. A biochemical function for
Ras at last. Science. 1994;264:1413-1414.
12. Treisman R. Ternary complex factors: growth factor regulated transcriptional activators. Curr Opin Genet Dev.. 1994;4:96-101.[Medline] [Order article via Infotrieve]
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