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(Hypertension. 1995;25:978-980.)
© 1995 American Heart Association, Inc.
Articles |
Na+-H+ Exchange and Essential Hypertension
A New Approach
From the Hypertension Research Center and the Department of Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark.
Correspondence to Dr Abraham Aviv, Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S Orange Ave, MSB F-464, Newark, NJ 07103-2714.
Key Words: hypertension • phosphorylation • calcium • phenotype • Na+-H+ exchange
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Introduction |
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 Top Introduction References |
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Enhanced activity of the ubiquitous (housekeeping) isoform of the Na+-H+ exchanger (NHE-1) is expressed in circulating cells from a subset of patients with essential hypertension (reviewed in Reference 11 ). This results from differences in pathways regulating exchanger function, since no mutations have been shown in the NHE-1 gene in patients with essential hypertension.2 3 A 1993 report by Rosskopf et al4 demonstrated that this phenotype persisted in immortalized lymphoblasts generated with Epstein-Barr virus from the lymphocytes of patients with essential hypertension. The lymphoblasts from hypertensive patients also showed an increased proliferation rate compared with those from normotensive subjects. Since these cells can be easily obtained from patients and maintained indefinitely in tissue culture, the findings of Rosskopf et al signaled the arrival of a new approach to studying the cellular manifestations of essential hypertension. In this issue of Hypertension, Ng and colleagues5 confirm the original findings of Rosskopf et al and provide important clues as to the molecular basis for the elevated NHE-1 activity in essential hypertension. These investigators show that the enhanced exchange activity is caused by an increased turnover of the exchanger itself and that this is associated with an increased phosphorylation of the NHE-1 protein. An additional finding reported by Ng et al is that the increased NHE-1 activity is also expressed in immortalized lymphoblasts from subjects who are normotensive but have a family history of essential hypertension.
There are several technical differences in the study of Ng et al5 and the previous study. Rosskopf et al4 selected hypertensive and control subjects primarily on the basis of previous determinations of Na+-H+ exchange activity in platelets, although family histories of essential hypertension were also considered. Ng et al relied on blood pressure measurements and family history to define the three subject groups, without prior determinations of Na+-H+ exchange activity in circulating cells. There were also technical differences in the assay procedures. Rosskopf et al measured Na+-H+ exchange activity in cells that had been treated for 10 minutes with a phorbol ester. This treatment produces an alkaline shift in the pH "set point" for activation of the exchanger, thereby increasing Na+-H+ exchange activity. In contrast, Ng et al used lymphoblasts that had been deprived of serum for 24 hours and did not treat the cells with a phorbol ester. Because phorbol esters stimulate protein kinase C, phosphorylation of the exchanger would probably be maximized under the conditions used by Rosskopf et al. Despite these differences, the results are qualitatively very similar. Both studies reported a highly significant increase in the apparent Vmax for Na+-H+ exchange activity and an increased proliferation rate in the cells from hypertensive subjects. Interestingly, Ng et al found that lymphoblasts from subjects with a family history of hypertension showed a higher Vmax for the Na+-H+ exchange but did not show an increased proliferation rate compared with lymphoblasts from control subjects. An important conclusion from both studies is that at least some of the factors that modulate Na+-H+ exchange activity and cellular proliferation rate in hypertensive subjects are expressed by the cells in tissue culture and therefore cannot be solely the result of altered levels of hormones or autacoids in the hypertensive individuals. Identifying the regulatory pathways that produce this phenotype in the immortalized lymphoblasts should therefore provide new insights into the intrinsic cellular alterations associated with essential hypertension. The NHE-1 protein provides a sharp molecular focus for such investigations.
Na+-H+ exchange activity is stimulated by or associated with a number of physiological variables, some of which are influenced by environmental factors; these include high salt intake,6 metabolic acidosis,7 hyperosmolarity,8 cellular spreading,9 and a host of growth factors and vasoactive agents10 11 12 (reviewed in References 11 , 1313 , and 1414 ). The regulatory pathways involved are very complex and have not been completely elucidated, although recent molecular studies of the NHE-1 protein have led to major advances in understanding its regulatory behavior (reviewed in Reference 1414 ). Na+-H+ exchangers (four mammalian isoforms have been described so far) consist of an N-terminal region of approximately 500 amino acids containing 10 to 12 membrane-spanning segments and a C-terminal hydrophilic domain of approximately 300 amino acids that lies on the cytosolic side of the membrane. The N-terminal hydrophobic domain is capable of carrying out Na+-H+ exchange in the absence of the C-terminal domain, but the latter is a regulatory region essential for mediating the response of the exchanger to growth factors and other activating stimuli. Exchange activity is increased by phosphorylation of NHE-1 at several serine residues in the C-terminal portion of the hydrophilic domain of the exchanger.15 The phosphatase inhibitor okadaic acid also stimulates exchange activity, and this is associated with increased exchanger phosphorylation.15 16 Changes in phosphorylation state are not the entire regulatory story, however, since deletion of the phosphorylation sites by site-directed mutagenesis only partially impairs the activation of exchange activity by growth factors.17 Moreover, contrary to expectations, treatment with protein kinase C inhibitors induced a further agonist-evoked alkaline shift in the pH set point.18 Interestingly, this increased shift is associated with a greater agonist-evoked rise in calcium.
The role of cytosolic calcium in regulating exchange activity has long been a controversial subject. However, recent studies provide unequivocal evidence that calmodulin is of central importance in regulating exchange activity.19 20 NHE-1 interacts with calcium/calmodulin at high- and low-affinity binding sites on the cytoplasmic regulatory domain. Mutational alterations in the exchanger that ablate the high-affinity calmodulin binding site also reduce or abolish the activation of exchange activity by growth factors or hyperosmolarity; the growth factor–induced increase in exchanger phosphorylation is not affected in these mutants, however. Thus, exchanger function is tied to two extremely complex and tightly controlled cellular regulatory pathways: the protein kinase/phosphatase cascade and cellular calcium homeostasis. Moreover, these pathways are closely linked, because protein kinases and phosphatases are intimately involved in calcium homeostasis and a number of protein kinases are themselves calcium-dependent enzymes.21
At yet another level of complexity, the exchanger has been shown to be activated by adhesion molecules such as fibronectin22 and to concentrate at focal adhesions, where it presumably interacts with cytoskeletal proteins. The effects of this interaction on exchange activity have yet to be precisely determined, although it is known that cellular spreading, a complex process involving integrins and cytoskeletal rearrangement, is associated with an increase in Na+-H+ exchange activity.9
The wealth of new information on the regulation of NHE-1 activity should facilitate mechanistic studies of its altered behavior in and its link to cellular proliferation in hypertensive subjects. Ng et al5 demonstrated an increased phosphorylation of the exchanger in lymphoblasts from hypertensive individuals. It is uncertain, however, whether this difference in phosphorylation state can fully account for the increased activity observed in the studies of Rosskopf et al,4 who used a phorbol ester to enhance Na+-H+ exchange activity. In fact, the increased exchange activity in untreated platelets from hypertensive versus normotensive subjects disappears when the platelets are treated with a phorbol ester.23 Nevertheless, these findings strongly suggest that the cells from hypertensive subjects display alterations in the protein kinase–phosphatase cascade that are directly or indirectly related to the increase in Na+-H+ exchange activity and the increased cellular proliferation rate. Additional factors are likely to play pivotal roles, including alterations in the calcium-calmodulin system, but these remain to be investigated. Studies of signaling pathways in these cells should generate important new information on the molecular and genetic mechanisms underlying the increased Na+-H+ exchange activity in essential hypertension and perhaps the pathogenesis of essential hypertension itself.
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Footnotes |
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The opinions expressed in this editorial comment 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. Rosskopf D, Dusing R, Siffert W. Membrane sodium-proton exchange and primary hypertension. Hypertension. 1993;21:607-617.
2.
Lifton RP, Hunt SC, Williams PR, Pouyssegur J, Lalouel
J-M. Exclusion of the Na+-H+ antiporter
as a candidate gene in human essential hypertension.
Hypertension. 1991;17:8-14.
3. Dudley CRK, Giuffra LA, Raine AEG, Reeders ST. Assessing the role of APNH, a gene encoding for a human amiloride-sensitive Na+/H+ antiporter, on interindividual variation in red cell Na+/Li+ countertransport. J Am Soc Nephrol. 1991;2:937-943. [Abstract]
4. Rosskopf D, Fromter E, Siffert W. Hypertensive sodium-proton exchanger phenotype persists in immortalized lymphoblasts from essential hypertensive patients: a cell culture model for human hypertension. J Clin Invest. 1993;92:2553-2559.
5.
Ng LL, Sweeney FP, Siczkowski M, Davies JE, Quinn PA,
Krolewski B, Krolewski AS. Na+-H+
antiporter phenotype, abundance and phosphorylation of immortalized
lymphoblasts from humans with hypertension.
Hypertension. 1995;25:971-977.
6. Gobel BO, Hoffmann G, Ruppert M, Stumpe KO, Vetter H, Siffert W, Dusing R. The lymphocyte Na+/H+ antiport: activation in primary hypertension and during chronic NaCl-loading. Eur J Clin Invest. 1994;24:529-539. [Medline] [Order article via Infotrieve]
7. Reusch HP, Reusch R, Rosskopf D, Siffert W, Mann JFE, Luft FC. Na+/H+ exchange in human lymphocytes and platelets in chronic and subacute metabolic acidosis. J Clin Invest. 1993;92:858-865.
8.
Grinstein S, Woodside M, Sardet C, Pouyssegur J, Rotin
D. Activation of Na+/H+
antiporter during cell volume regulation: evidence for a
phosphorylation-independent mechanism. J Biol Chem. 1992;267:23823-23828.
9.
Schwartz MA, Both G, Lechene C. Effect of cell
spreading on cytoplasmic pH in normal and transformed
fibroblasts. Proc Natl Acad Sci U S A. 1989;86:4525-4529.
10.
Sardet C, Counillon L, Franchi A, Pouyssegur J.
Growth factors induce phosphorylation of the
Na+/H+ antiporter, a glycoprotein of 110
kD. Science. 1990;247:723-726.
11. Kimura M, Lasker N, Aviv A. Cyclic nucleotides attenuate thrombin-evoked alterations in parameters of platelet N/H antiport: the role of cytosolic Ca. J Clin Invest. 1992;89:1121-1127.
12.
Hatori N, Fine BP, Nakamura A, Cragoe E Jr, Aviv A.
Angiotensin II effect on cytosolic pH in cultured rat vascular
smooth muscle cells. J Biol Chem. 1987;262:5073-5078.
13. Wakabayashi S, Sardet C, Fafournoux P, Counillon L, Meloche S, Pages G, Pouyssegur J. Structure function of the growth factor-activated Na+/H+ exchanger (NHE-1). Rev Physiol Biochem Pharmacol. 1992;119:157-186. [Medline] [Order article via Infotrieve]
14.
Bianchini L, Pouyssegur J. Molecular structure
and regulation of vertebrate Na+/H+
exchangers. J Exp Biol. 1994;196:337-345.
15.
Sardet C, Fafournoux P, Pouyssegur J.
-Thrombin, epidermal growth factor, and okadaic acid activate
the Na+/H+ exchanger, NHE-1, by
phosphorylating a set of common sites. J Biol Chem. 1991;266:19166-19171.
16.
Bianchini L, Woodside M, Sardet C, Pouyssegur J, Takai
A, Grinstein S. Okadaic acid, a phosphatase inhibitor, induces
activation and phosphorylation of the
Na+/H+ antiport. J Biol
Chem. 1991;266:15406-15413.
17.
Wakabayashi S, Bertrand B, Shigekawa M, Fafournoux P,
Pouyssegur J. Growth factor activation and
`H+-sensing' of the
Na+/H+
exchanger isoform 1 (NHE1): evidence for an additional mechanism not
requiring direct phosphorylation. J Biol Chem. 1994;269:5583-5588.
18.
Kimura M, Gardner J, Aviv A. Agonist-evoked
alkaline shift in the cytosolic pH setpoint for activation of
Na+/H+ antiport in human platelets: the
role of cytosolic Ca2+ and protein kinase C.
J Biol Chem. 1990;265:21068-21074.
19.
Bertrand B, Wakabayashi S, Ikeda T, Pouyssegur J,
Shigekawa M. The Na+/H+ exchanger
isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins:
identification and characterization of calmodulin binding
sites. J Biol Chem. 1994;269:13703-13709.
20.
Wakabayashi S, Bertrand B, Ikeda T, Pouyssegur J,
Shigekawa M. Mutation of calmodulin-binding site renders the
Na+/H+ exchanger (NHE1) highly
H+-sensitive and Ca2+ regulation
defective. J Biol Chem. 1994;269:13710-13715.
21. Parker PJ, Kour G, Marais RM, Mitchell F, Pears C, Schaap D, Stabel S, Webster C. Protein kinase C: a family affair. Mol Cell Endocrinol. 1989;65:1-11. [Medline] [Order article via Infotrieve]
22.
Schwartz MA, Lechene C, Ingber DE. Insoluble
fibronectin activates the Na/H antiporter by clustering and
immobilizing integrin 5ß1 independent of cell
shape. Proc Natl Acad Sci U S A. 1991;88:7849-7853.
23. Livne AA, Aharonovitz O, Paran E. Higher Na+-H+ exchange rate and more alkaline intracellular pH set-point in essential hypertension: effects of protein kinase modulation in platelets. J Hypertens. 1991;9:1013-1019. [Medline] [Order article via Infotrieve]
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