© 2001 American Heart Association, Inc.
Editorials |
EDHF and NO
Different Pathways for Production—Similar Actions
From the Department of Physiology and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wis.
Correspondence to William M. Chilian, PhD, Department of Physiology, Cardiovascular Center, 8701 Watertown Plank Rd, Milwaukee, WI 53226-0509. E-mail chilian{at}mcw.edu
Key Words: nitric oxide • endothelium-derived hyperpolarizing factor • vasodilation • endothelium
In this issue of Circulation Research, Véquaud and Thorin1 present observations that attest to differential G protein signaling in dilation to nitric oxide (NO) and endothelium-derived hyperpolarization factor (EDHF). The investigators also report that EDHF of the mesenteric artery is not a metabolite of phospholipase C, which strongly suggests that this vasodilator is not a lipid metabolite of cytochrome P450. Véquaud and Thorin also reported that EDHF-mediated dilation is calcium independent, which contrasts to the calcium dependency of agonist-induced NO-mediated dilation. And finally the authors report that heat shock protein 90 (Hsp90) participates in the production of NO but not EDHF. This study not only provides important contributions to our understanding of NO- and EDHF-mediated vasodilation but also presents a methodological tour de force in the study of microvascular signaling using the intracellular application of antibodies as specific inhibitors of the signaling pathways.
With regard to the methodological advance, the authors’ method for the study of signaling pathways in intact microvessels is novel and is based on a technique described to study transduction pathways in cultured cells.2 Specifically, antibodies to specific G proteins or Hsp90 were incorporated into the endothelium of isolated pressurized microvessels using osmotic shock—hyperosmotic conditions followed by hypoosmotic shock. After this treatment, the nonspecific effects appeared nonexistent as evidenced by restoration of spontaneous tone and myogenic responses. The advantage of this approach is that the effects of an antibody should be specific. This contrasts to the application of so-called "specific" inhibitors of enzymes—most of these inhibitors are not specific, and preferential is a better descriptive term. Although many inhibitors have reported Ki values, it is very difficult to estimate the intracellular concentrations in systems as simple as cultured monolayers of cells, and even more problematic in cells in isolated vessels in organ chambers. The use of antibodies to probe signaling pathways represents a truly novel approach to study signaling in intact blood vessels and microvessels and circumvents many of the problems related to specificity with pharmacological inhibitors.
The observation that Hsp90 is required for endothelial NO synthase (eNOS) function is well documented by several laboratories,3,4 and the present observations confirm these reports.1 The novel aspect of these particular findings is that Hsp90 is not required for the synthesis and/or actions of EDHF.
Véquaud and Thorin1 also reported that EDHF is not a metabolite of phospholipase C and does not require increases in intracellular calcium for production. Although these data do not reveal the identity of EDHF in the mesenteric artery, they suggest what it is not. Along this line, it is worth mentioning that the identity of EDHF is controversial, and our opinion for the basis of this controversy relates to the likelihood that there are many EDHFs. Some groups report that EDHF in coronary arteries and arterioles is a lipid metabolite of cytochrome P450,5–7 and the general presumption is that the lipid substrate for cytochrome P450 is produced by the actions of phospholipase C on membrane phospholipids. However, several other groups have reported that EDHF is not a metabolite of cytochrome P450, and the identity has been suggested as enandimides, K+, or other fatty acid metabolites.8–11 The authors also observed that Ca2+ signaling is not involved in the production of EDHF, which contrasts greatly to the signal cascade activated during agonist-induced production of NO. Despite their compelling results, a caveat should be mentioned: with the probable existence of many EDHFs, one should not necessarily conclude that Ca2+ is not involved, because in another EDHF, this cation may hold a seminal role.
An important aspect of the results presented by Véquaud and Thorin1 relates to the differential G protein signaling involved in the production of NO and EDHF. In a similar vein, one should also exercise caution regarding a general conclusion about the involvement of G proteins in all EDHFs. Because of the many organ system and species differences that seem to underscore the many faces of EDHF, a universal involvement of a certain G protein in the production of this vasodilator seems unlikely. Despite this caveat, the authors’ results that G protein 
-subunits and ß-subunits are involved in NO- and EDHF-mediated vasodilation, respectively, are important. This conclusion, and advance, was revealed by administration of the specific antibody against the particular G-protein subunit to the endothelium of the intact vessel. Because the antibody was administered intraluminally, the effect is largely confined to endothelial cells. This is important to highlight because it engendered the authors to discriminate between the production of the vasodilator versus the actions of the substance. The results also imply a level of discrete regulation of the production of EDHF and NO. Moreover, the existence of discrete signaling pathways to produce vasodilation may be underscored by a necessity to evolve parallel or redundant controls. Such a system would have the safeguards of backup controls, which may be important in the event one of the dilator pathways is compromised.
In the aggregate, the observations that EDHF is distinct from NO would appear to confer some benefits to vascular control mechanisms. Sir Isaac Newton once stated, "Nature does not believe in the pomp of superfluous causes," which in the context of the cardiovascular system implies that systems exist because they offer an advantage in the regulation of blood flow and vasomotor tone. Having both NO and EDHF as regulatory systems is most likely important. If one particular system fails, then the parallel or backup system could be activated to assume vasomotor control. Such interactions between NO and EDHF have been observed previously.12,13 As the report by Véquaud and Thorin1 demonstrates, EDHF and NO produce a similar net effect, ie, vasodilation, but are distinct insofar as they are produced by completely different signaling cascades. This scheme for vasomotor control would appear advantageous, because parallel or backup controls would be produced by different transduction pathways and would likely have different downstream biochemical effectors. Thus, the coexistence of distinctive transduction pathways for the production of NO and EDHF would appear to confer flexibility and safeguards in the control of organ blood flow under a variety of physiological and pathophysiological conditions.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
1.
Véquaud P, Thorin E. Endothelial G protein ß-subunits trigger nitric oxide– but not endothelium-derived hyperpolarizing factor–dependent dilation in rabbit resistance arteries. Circ Res. 2001; 89: 716–722.
2. Okada CY, Rechsteiner M. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell. 1982; 29: 33–41.[Medline] [Order article via Infotrieve]
3. Garcia-Gardena G, Ran R, Shah V, Sorrentino R, Cirinos G, Papapetropopoulos A, Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp 90. Nature. 1998; 392: 821–824.[Medline] [Order article via Infotrieve]
4.
Pritchard KA Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Fontana JT, Baker JE, Sessa WC. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric oxide synthase. J Biol Chem. 2001; 276: 17621–17624.
5.
Li PL, Campbell WB. Epoxyeicosatrienoic acids activate K+ channels in coronary smooth muscle through a guanine nucleotide binding protein. Circ Res. 1997; 80: 877–884.
6. Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, Busse R. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature. 1999; 401: 493–497.[Medline] [Order article via Infotrieve]
7.
Nishikawa Y, Stepp DW, Chilian WM. In vivo location and mechanism of EDHF-mediated vasodilation in canine coronary microcirculation. Am J Physiol. 1999; 277: H1252–H1259.
8. Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature. 1998; 396: 269–272.[Medline] [Order article via Infotrieve]
9.
Brandes RP, Schmita-Winnenthal FH, Feletou M, Goedecke A, Huang PL, Vanhoutte PM, Fleming I, Busse R. An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice. Proc Natl Acad Sci USA. 2000; 97: 9747–9752.
10.
Pfister SL, Spitzbarth N, Edgemond W, Campbell WR. Vasorelaxation by an endothelium-derived metabolite of arachidonic acid. Am J Physiol. 1996; 270: H1021–H1030.
11. Randall MD, Alexander SPH, Bennett T, Boyd EA, Fry JR, Gardiner SM, Kemp PA, McCulloch AI, Kendall DA. An endogenous cannabinoid as an endothelium-dependent vasorelaxant. Biochem Biophys Res Commun. 1996; 229: 114–120.[Medline] [Order article via Infotrieve]
12.
Bauersachs J, Popp R, Hecker M, Sauer E, Fleming I, Busse R. Nitric oxide attenuates the release of endothelium-derived hyperpolarizing factor. Circulation. 1996; 94: 3341–3347.
13.
Nishikawa Y, Stepp DW, Chilian WM. Nitric oxide exerts feed back inhibition on EDHF-induced coronary arteriolar dilation in vivo. Am J Physiol. 2000; 279: H459–H465.
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