INVITED REVIEW
Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

¹ Laboratory of Molecular Medicine, Centre de Recherche de L'Université de Montreal, Campus Hotel-Dieu, Montreal, Quebec, Canada; ² Laboratory of Biomembranes, Faculty of Biology, Moscow State University, Moscow, Russia; and ³ Department of Pharmacology and Toxicology, Wright State University, Dayton, Ohio

	ABSTRACT

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

Data obtained during the last two decades show that spontaneously hypertensive rats, an acceptable experimental model of primary human hypertension, possess increased activity of both ubiquitous and renal cell-specific isoforms of the Na⁺/H⁺ exchanger (NHE) and Na⁺-K⁺-2Cl cotransporter. Abnormalities of these ion transporters have been found in patients suffering from essential hypertension. Recent genetic studies demonstrate that genes encoding the - and -subunits of ENaC, a renal cell-specific isoform of the Na⁺-K⁺-2Cl cotransporter, and 3-, 1-, and 2-subunits of the Na⁺-K⁺ pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis of the homology of genome maps, several other genes encoding these transporters, as well as the Na⁺/H⁺ exchanger and Na⁺-K⁺-2Cl cotransporter, can be predicted in QTL related to the pathogenesis of hypertension. However, despite their location within QTL, analysis of cDNA structure did not reveal any mutation in the coding region of the above-listed transporters in primary hypertension, with the exception of G276L substitution in the 1-Na⁺-K⁺ pump from Dahl salt-sensitive rats and a higher occurrence of T594M mutation of -ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities of systems involved in the regulation of their expression and/or function. Further analysis of QTL in F₂ hybrids of normotensive and hypertensive rats and in affected sibling pairs will allow mapping of genes causing abnormalities of these regulatory pathways.

ion transporters; genes; vascular and renal function

	INTRODUCTION

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

IN THE MID-1970's, it was reported that the ouabain-insensitive component of monovalent ion exchange was increased in aortic strips from spontaneously hypertensive rats (SHR) (133) as well as in erythrocytes from SHR and patients with essential hypertension (231, 232). Later on, these results were confirmed in several laboratories and used as a basis to assess the involvement of abnormal membrane ion transport in the pathogenesis of primary hypertension (229, 230). During the last two decades, these abnormalities were shown to be caused by altered function of carriers implicated in monovalent ion transport, such as Na⁺/H⁺ exchange, Na⁺/Li⁺ (Na⁺/Na⁺) countertransport, Cl-dependent Na⁺-K⁺ cotransport, and of the Na⁺-K⁺ pump as well as Na⁺ and K⁺ channels. The first part of the present review summarizes our current knowledge on the activity of these ion transport systems in primary hypertension. Recent progress in molecular biology and the genetics of complex disorders led to cloning of the above-listed ion transporters and identification of gene loci for elevated blood pressure and its complications. These findings enabled us to analyze the possible molecular and genetic determinants of abnormal ion transport in primary hypertension and their involvement in the pathogenesis of this disease.

	IDENTIFICATION OF ALTERED MONOVALENT ION TRANSPORTERS IN PRIMARY HYPERTENSION

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

Na⁺/H⁺ Exchange

Na⁺/H⁺ exchange provides electroneutral countertransport of Na⁺ (Li⁺) and H⁺ and is involved in the regulation of cell volume as well as intracellular concentrations of Na⁺ and H⁺. In epithelial cells, this ion carrier is also involved in transcellular movement of salt and osmotically obliged water. The cDNA encoding this carrier was first cloned from a human genomic DNA library, using the so-called H⁺ suicide strategy in a Na⁺/H⁺ exchange-deficient fibroblast line (252). mRNA probes showed that this form of transporter, referred to as housekeeping or ubiquitous Na⁺/H⁺ exchanger type 1 (NHE1), was expressed in all mammalian cells studied so far. Subsequently, the tissue-specific forms of the Na⁺/H⁺ exchanger (NHE2 to NHE6) derived from different genes were revealed by low-stringency screening of cDNA libraries with NHE1 cDNA or related oligonucleotide probes. Apart from differences in tissue expression, the cloned forms of the Na⁺/H⁺ exchanger possess different sensitivities to amiloride and its derivatives, as well as to the modulatory effect of 4-phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, and cell volume (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Properties of Na+/H+ exchanger isoforms (30, 31, 38, 138, 185, 209, 289, 296)

The nucleotide sequence of NHE1 cDNA, possessing high homology in mammalian species (>90%), predicts a protein of ~91 kDa with a deduced secondary structure consisting of a short cytoplasmic NH₂ terminus, 12 transmembrane domains, and an extended hydrophilic COOH terminus. All cloned forms of the Na⁺/H⁺ exchanger share these features of secondary structure of NHE1 with 60% homology in the sequence encoding a region from the 2nd to the 12th transmembrane domain and 30% homology in the COOH-terminal region. Complete removal of the NHE1 COOH terminus preserves allosteric activation of the exchanger by intracellular H⁺ with a Hill coefficient >2 but shifts pH dependence to a more acidic intracellular pH and completely abolishes the regulation of NHE1 by growth factors as well as by hormones and neurotransmitters coupled to seven membrane-domain-spanning receptors. The same results have been obtained for epithelial cell-specific forms of the Na⁺/H⁺ exchanger (for more details, see Refs. 162, 289, 296).

With the exception of a few cell types, the activity of the Na⁺/H⁺ exchanger is quenched under basal conditions. Data on the functional properties of this carrier in primary hypertension have been mainly obtained from the study of Na⁺-dependent amiloride-sensitive H⁺ fluxes or amiloride-sensitive Na⁺ uptake in Na⁺-depleted cells with a prior acidified cytoplasm (240, 264). With this approach, electrochemical H⁺ gradient (µ_H⁺)-induced Na⁺/H⁺ exchange activity is increased in platelets, lymphocytes, neutrophils, erythrocytes, mesangial cells, kidney epithelial cells, freshly isolated segments of arteries, cultured vascular smooth muscle cells (VSMC), and striated muscle cells from SHR as well as in erythrocytes, platelets, lymphocytes, and immortalized lymphoblasts from patients with essential hypertension. In vivo studies using NMR spectroscopy have revealed augmented activity of the Na⁺/H⁺ exchanger in exercising skeletal muscle of SHR and essential hypertensive patients (Table 2). Table 3 shows that this difference is caused by increased maximal activity of the Na⁺/H⁺ exchanger rather than its sensitivity to intracellular H⁺.

View this table: [in this window] [in a new window]   Table 2.   Activity of Na+/H+ exchanger in primary hypertension

View this table: [in this window] [in a new window]   Table 3.   Kinetic properties of Na+/H+ exchange in primary hypertension

In contrast to SHR, using the same experimental approach, we failed to detect any modification of this carrier's activity in erythrocytes and kidney epithelial cells from a colony of spontaneously hypertensive rats developed in Milan [Milan hypertensive strain (MHS); Table 2]. Based on a comparative analysis of abnormalities of this and other ion transporters (see below), it has been claimed that different strains of rodents with genetically determined hypertension can be employed for the study of different aspects of the pathogenesis of essential hypertension arising as a result of complex interactions of different genetic determinants and the environment (72). Indeed, in accordance with this mosaic theory of the pathogenesis of essential hypertension originally proposed by Page (211), an analysis of Na⁺/H⁺ exchange activity in erythrocytes from normotensive and hypertensive patients demonstrated that only 40-50% of subjects with essential hypertension possess increased Na⁺/H⁺ exchange activity similar to that seen in SHR, whereas, in the remaining essential hypertensive patients, this transporter is unaltered, as observed in MHS (27, 77).

Aside from primary hypertension, augmented Na⁺/H⁺ exchange activity has also been reported in blood cells, immortalized lymphoblasts, and skin fibroblasts from insulin-dependent diabetes mellitus (IDDM) patients with nephropathy (for recent review, see Ref. 286). It is well documented that hypertension and predisposition to it are essential components in the pathogenesis of IDDM. The mechanisms underlying this linkage are poorly understood. It is possible that enhanced Na⁺/H⁺ exchange activity is a common element in the pathogenesis of these diseases.

Based on the ubiquitous expression of NHE1 (Table 1), this isoform is assumed to be mainly involved in the heightened Na⁺/H⁺ exchange activity seen in blood cells and myocytes of SHR and patients with essential hypertension (Table 2). This conclusion is also supported by data on the lack of expression of epithelial cell-specific NHE2-NHE4 in SHR VSMC possessing enhanced Na⁺/H⁺ exchange activity (170). In the early 1990's, we reported data on increased Na⁺/H⁺ exchange activity in primary cultured renal epithelial cells from SHR (200). Simultaneously with our observation, an augmented rate of Na⁺/H⁺ exchange was demonstrated in isolated proximal tubules from SHR (43, 86). However, these studies did not evaluate the relative contribution of basolateral and renal cell-specific apical Na⁺/H⁺ exchanger in these abnormalities. Recently, using a novel, highly specific inhibitor of NHE1, HOE-694, it was shown that both NHE1 and NHE3 activities are increased in SHR proximal tubule cells (118, 140). Viewing these results in conjunction with data on the increased rate of amiloride-sensitive ²²Na⁺ uptake in brush-border membrane vesicles from proximal tubules of MHS (218), it may be assumed that, in contrast to NHE1, both SHR and MHS exhibit enhanced NHE3 activity.

Can We Use Erythrocyte Na⁺/Li⁺ Countertransport as a Marker of NHE1 Activity?

In the mid-1970's, it was reported that the rate of Li⁺ efflux from human erythrocytes could be diminished by two- to threefold under isosmotic substitution of extracellular Na⁺ with Mg²⁺ and sucrose. Keeping in mind that Li⁺ substitutes for Na⁺ in most of the transport systems studied so far, the extracellular Na⁺-dependent component of Li⁺ efflux, termed Na⁺/Li⁺ exchange or countertransport, was assumed to represent a mode of operation of the equimolar Na⁺/Na⁺ exchanger (59, 216, 253). In 1980, we demonstrated that the rate of Na⁺/Li⁺ countertransport is increased in erythrocytes from patients with essential hypertension (29). During the last 15 years, this observation was reproduced in many laboratories (for review see Refs. 109, 114, 125, 230). Both enhanced maximal activity and apparent affinity to extracellular Na⁺ contribute to the enhanced rate of erythrocyte Na⁺/Li⁺ countertransport in essential hypertension (248, 257). For more details, see Ref. 114.

Despite abundant (in >500 papers) data on augmented Na⁺/Li⁺ countertransport in essential hypertension, the molecular mechanism of this phenomenon is still unknown. The lack of a selective inhibitor of this carrier complicates its purification by affinity chromatography and identification by an expression/cloning strategy. An attractive hypothesis suggests that enhanced erythrocyte Na⁺/Li⁺ countertransport is a marker of increased activity of the housekeeping or renal cell-specific isoform of the Na⁺/H⁺ exchanger (5, 125). However, detailed comparison of the properties of erythrocyte Na⁺/Li⁺ countertransport and µ_H⁺induced Na⁺ and H⁺ fluxes in erythrocytes and in cells transfected with the cloned isoform of the Na⁺/H⁺ exchanger argues against this hypothesis. Thus it has been shown that ATP depletion does not modify the activity of Na⁺/Li⁺ countertransport in human erythrocytes but markedly inhibits erythrocyte µ_H⁺-induced Na⁺/H⁺ exchange as well as the activity of all cloned Na⁺/H⁺ exchangers. One millimolar amiloride completely suppresses the activities of NHE1 and NHE2 and partly suppresses the activities of NHE3 and NHE4 (Table 1) but does not affect erythrocyte Na⁺/Li⁺ countertransport. Cell shrinkage and PMA, the activator of protein kinase C, are well-documented modulators of the activity of cloned forms of the Na⁺/H⁺ exchanger (Table 1). However, these modulators do not affect the activity of erythrocyte Na⁺/Li⁺ countertransport. The most striking results were obtained by comparison of the relative activity of Na⁺/Li⁺ countertransport and µ_H⁺-induced Na⁺/H⁺ exchange in erythrocytes of different species. Thus we failed to demonstrate Na⁺/Li⁺ countertransport in rat erythrocytes, whereas µ_H⁺-induced Na⁺/H⁺ exchange in these cells was four- to fivefold higher compared with human erythrocytes. In rabbit erythrocytes, Na⁺/Li⁺ countertransport is 20-fold higher compared with human cells, whereas the activity of µ_H⁺-induced Na⁺/H⁺ exchange in these species is about the same (for a more detailed comparison of the properties of erythrocyte Na⁺/Li⁺ countertransport, µ_H⁺-induced Na⁺/H⁺ exchange, and cloned forms of the Na⁺/H⁺ exchanger, see Ref. 193). These results show that erythrocyte Na⁺/Li⁺ countertransport cannot be used as a marker of enhanced NHE1 activity in essential hypertension. Further experiments should clarify whether these carriers are encoded by distinct genes or whether the special properties of the erythrocyte Na⁺/Li⁺ countertransporter are caused by posttranslational modification of NHE1-NHE4 gene products during red blood cell maturation.

Cl-Dependent Cotransporters

In the late 1970's, it became clear that coupled fluxes of Cl, Na⁺, and K⁺ across the plasma membrane are maintained by ion carriers, providing Cl-dependent electroneutral cotransport (uniport) of monovalent ions (160). During the last few years, cDNAs encoding Cl-dependent cotransporters were isolated using the expression-cloning strategy (Table 4). All members of the superfamily of Cl-dependent cotransporters possess about the same relative molecular mass (M_r) of ~110-130 kDa and the same membrane architecture consisting of 12 transmembrane domains and large COOH and NH₂ cytoplasmic terminals. Two members of this superfamily (NKCC1 and NKCC2), which operate only under the simultaneous presence of Na⁺, K⁺, and Cl, are completely blocked by p-sulfamoylbenzoic acid derivatives, such as furosemide, and its more selective analog bumetanide. In accordance with early data, both ubiquitous (NKCC1) and renal cell-specific (NKCC2) forms of the furosemide-sensitive carrier function as Na⁺-K⁺-2Cl cotransport (84, 91). However, later on, it became clear that these carriers can also operate as 1Na⁺-2K⁺-3Cl cotransport, K⁺-dependent Na⁺-Cl cotransport, Na⁺-dependent K⁺-Cl cotransport, (Na⁺ + Cl)-dependent K⁺/K⁺ exchanger and (K⁺ + Cl)-dependent Na⁺/Na⁺ exchanger. Moreover, the mode of operation of these carriers is flexible and varies under hormonal activation and cell volume alteration (101, 206). In most cells, NKCC1 and NKCC2 are activated by cell shrinkage and inhibited by swelling. In human erythrocytes, furosemide-sensitive K⁺ fluxes are insensitive to isosmotic and hyperosmotic cell shrinkage (2, 198), whereas in astrocytes (178), Ehrlich ascites tumor cells (159), and C6 glioma cells (177) this ion transport pathway is activated by cell swelling. The relative contribution of Na⁺-independent K⁺-Cl cotransporters in this phenomenon remains unknown. The other member of the superfamily, NCC, encoding the K⁺-independent, furosemide-resistant, thiazide-sensitive Na⁺-Cl cotransporter, has been found in epithelial cells derived from distal tubules. Both the ubiquitous and brain-specific isoforms of Na⁺-independent K⁺-Cl cotransporters (KCC1 and KCC2, respectively) are much less selective to furosemide compared with NKCC. In all types of cells studied so far, KCC1 is activated by cell swelling and inhibited by cell shrinkage (161). To the best of our knowledge, there are no data on volume-dependent regulation of NCC and KCC2.

View this table: [in this window] [in a new window]   Table 4.   Properties of Cl-dependent cotransporters (9, 47, 80, 87, 101, 137, 173, 220-222)

The first indication of altered activity of the ubiquitous form of the Na⁺-K⁺-2Cl cotransporter (NKCC1) in primary hypertension was obtained by Garay with co-workers in a study of furosemide-sensitive ion transport in erythrocytes from SHR and patients with essential hypertension (50, 81). These results, however, were obtained in cells treated with sulfhydryl reagents and with inverse transmembrane gradients of Na⁺ and K⁺ that complicate their implication for analysis of the activity of this carrier in vivo. Data on the activity of this carrier in untreated cells are summarized in Table 5. The outward mode of operation of Na⁺-K⁺-2Cl cotransport measured as the rate of furosemide/bumetanide-sensitive Na⁺ or K⁺ (Rb⁺) efflux was reported to be increased in erythrocytes from SHR and MHS by ~30 and ~50%, respectively. The negative result obtained by Yokomatsu and co-workers (302) was probably caused by overestimation of the rate of ²²Na⁺ efflux at high internal Na⁺ in erythrocytes from SHR. The importance of backflux correction to compare the outward mode of operation of ion transporters has been discussed previously in detail (228). In the case of Na⁺-K⁺-2Cl cotransport, this fact is especially important because of the inhibition of its activity by internal Cl and Na⁺ (19, 101) and of the increased sensitivity for intracellular Na⁺ in erythrocytes from MHS (73). Both enhanced and unaltered Na⁺-K⁺-2Cl cotransport activities have been reported in studies on the outward and inward modes of operation of this carrier in erythrocytes from patients with essential hypertension (Table 5). This discrepancy can be explained by the presence of endogenous circulating inhibitors of Na⁺-K⁺-2Cl cotransport (272) that can affect the activity of the carrier in freshly isolated blood cells. It should be noted that, in contrast to the Na⁺/H⁺ exchanger, which is enhanced in about one-half of the essential hypertensive population (see Na⁺/H⁺ Exchange), Na⁺-K⁺-2Cl cotransport activity is increased in only 20-25% of essential hypertensive subjects possessing low plasma renin activity (24, 41, 42).

View this table: [in this window] [in a new window]   Table 5.   Activity of Na+-K+-2Cl cotransport in primary hypertension

The data on Na⁺-K⁺-2Cl cotransport in nucleated cells from SHR are rather contradictory. Thus the activity of this carrier has been reported to be increased, decreased, or unaltered in VSMC from SHR (Table 5). For an analysis of this discrepancy, it is important to underline that, in contrast to mature mammalian erythrocytes, the permeability of nucleated cells for Na⁺ is extremely high, and Na⁺-K⁺-2Cl cotransport activity may be altered due to the uncontrolled modification of intracellular ion content caused by different ionic composition of the medium and by treatment with ouabain. Na⁺-K⁺-2Cl cotransport seems to be also sensitive to the stage of cell cycle progression. Thus Raat and colleagues (237) reported that Na⁺-K⁺-2Cl cotransport is active in cells growing in culture but is quenched in freshly isolated rat VSMC, endothelial cells, and rabbit proximal tubules. However, Yerby et al. (301) observed active Na⁺-K⁺-2Cl cotransport in freshly isolated endothelial cells. This point needs further clarification.

According to data on the tissue distribution of Na⁺-K⁺-2Cl cotransport isoforms (Table 4), an enhanced rate of bumetanide-sensitive ion flux in nonepithelial cells is caused by hyperactivity of NKCC1. Data on Na⁺-K⁺-2Cl cotransport activity in epithelial cells are limited to a few publications. It has been shown that the diuretic response to furosemide is exaggerated in perfused kidney from MHS (249). The same research team has reported that erythrocyte Na⁺-K⁺-2Cl cotransport activity in hypertensive patients is highly and positively correlated with Na⁺ loss caused by furosemide administration (42, 238). Table 5 shows that Na⁺-K⁺-2Cl cotransport is increased in cultured tubular epithelial cells and in membrane fractions from this nephron segment of MHS. However, the relative contribution of basolateral NKCC1 and apical NKCC2 to this abnormality is still unknown.

NCC activity in primary hypertension has not yet been explored. There is no indication of modification of bumetanide-resistant K⁺ fluxes in erythrocytes from SHR and patients with essential hypertension (202, 305) as well as in SHR VSMC (151, 208), which suggests that K⁺-Cl cotransport activity is not altered in primary hypertension. In contrast to K⁺, the rate of ouabain-plus-bumetanide-insensitive Na⁺ fluxes is slightly increased in SHR erythrocytes (304). However, it is still not clear whether these differences are caused by altered passive permeability of the plasma membrane for this cation (membrane leakage) or caused by the presence of a nonidentified ion carrier. The impact of increased surface area in a fixed volume of packed cells due to diminished volume of erythrocytes from SHR compared with normotensive Wistar-Kyoto rats (WKY) (225, 227) should also be considered when these data are analyzed.

Amiloride-Sensitive Na⁺ Channels

Amiloride-sensitive Na⁺ channels provide selective movement of Na⁺ along its electrochemical gradient. These channels are more sensitive to amiloride than is NHE1 and are highly resistant to other potent inhibitors of the Na⁺/H⁺ exchanger, such as ethylisopropylamiloride (EIPA). Unlike neuronal TTX-sensitive Na⁺ channels, amiloride-sensitive channels do not show voltage-dependent gating. Channels possessing these properties are mainly expressed in the apical membrane of epithelial cells and are called epithelial Na⁺ channels (ENaC; for more details, see Refs. 83 and 215). Three homologous subunits of ENaC with M_r 72-79 kDa and classified as , , and  were cloned from the rat distal colon by functional expression in Xenopus oocytes (25, 26). It was shown that the -subunit possesses amiloride-sensitive Na⁺ conductance, whereas the - and -subunits augment the channel's activity. Hydrophobicity plot analysis indicates that all ENaC subunits have two membrane-spanning domains linked by an ~500-amino-acid extracellular loop and flanked by cytoplasmic NH₂ and COOH terminals. The COOH terminus contains two proline-rich segments that resemble the SH3-binding domain. These domains probably play a key role in the formation of an active heterotrimer and more active 3/3/3 (271) or 2// (75, 143) complex. An additional cDNA, defined as the -subunit sharing ~40% homology with the -subunit and 25-30% homology with the - and -subunits, has been cloned by cDNA library screening (297). -ENaC was revealed in the majority of nonepithelial cells studied so far with extremely low expression in the kidney. Similar to -ENaC, the amiloride-sensitive conductance of nonepithelial -ENaC is sharply increased by coexpression with the - and -subunits (83).

In contrast to the monogenic forms of low-renin hypertension (see Search for mutations), data on the enhanced activity of ENaC in primary hypertension are limited to a few observations. In epithelial monolayers derived from the inner medullary collecting duct, cells from salt-sensitive Dahl rats (SS/JR) transport twice as much Na⁺ as cells from their salt-resistant counterparts (SR/JR) (126). On the basis of electrophysiological data, it was concluded that this difference may be caused by enhanced activity of the basolateral Na⁺-K⁺ pump or apical ENaC. Neither the maximal activity of the Na⁺-K⁺ pump nor its apparent affinity for intracellular Na⁺ and extracellular K⁺ was altered in salt-sensitive rats, suggesting higher Na⁺ transport via ENaC (127). Several types of nonepithelial cells including lymphoid cells also exhibit an amiloride-sensitive component of Na⁺ conductance (22). Bubien et al. (21) studied the electrophysiological properties of lymphocytes from 20 untreated patients possessing severe hypertension regardless of treatment with different classes of antihypertensive drugs. In this study, 14 patients were characterized by constitutively activated, amiloride-sensitive Na⁺ channels, whereas in other patients these channels appeared after elevation of intracellular cAMP content only. Interestingly, treatment with amiloride of patients possessing constitutively activated Na⁺ channels led to significant reduction of blood pressure (21). This finding should be further examined in more representative population studies and under comprehensive comparative analysis of the properties that amiloride-sensitive Na⁺ channels expressed in renal epithelium and in white blood cells.

Na⁺-K⁺ Pump

The Na⁺-K⁺ pump provides ouabain-sensitive hydrolysis of ATP coupled to the inward movement of K⁺ and outward movement of Na⁺, playing a key role in regulation of the intracellular concentration of monovalent cations. Because of its electrogenicity (3Na⁺/2K⁺), the Na⁺-K⁺ pump also contributes to the regulation of membrane potential in electrically excitable cells and other cell types with relatively high plasma membrane electrical resistance. The minimal functional unit of the Na⁺-K⁺ pump is composed of - and -subunits assembled in a 1:1 ratio. Three isoforms of - and -subunits have been cloned up to now. The -subunit has 10 transmembrane domains with both NH₂ and COOH endoplasmic terminals. All -subunits contain ATP, Na⁺, K⁺, and ouabain-binding sites, exhibit Mg²⁺-dependent ATP hydrolysis, and are able to provide movements of Na⁺ and K⁺ against their electrochemical gradients. The major differences between isoforms of -subunits are related to tissue distribution and their sensitivity to activation by intracellular Na⁺ and inhibition by ouabain [half-maximal activation (K_0.5) for intracellular Na⁺: 12, 24, and 23 mM; half-maximal inhibition (K_i) for ouabain: >10, 0.02-0.05, and <0.01 µM for 1-, 2-, and 3-subunits, respectively (85, 119, 260)]. The -subunit consists of a short cytoplasmic NH₂ terminus, one transmembrane domain, and a highly glycosylated extended ectodomain. It is involved in regulation of the affinity of the enzyme for extracellular K⁺ and for reassembly of the Na⁺-K⁺ pump within the plasma membrane (for more details, see Refs. 65, 85, 290).

Intensive studies of erythrocyte Na⁺/K⁺-ATPase activity in primary hypertension, performed in the late 1970's and early 1980's, did not reveal any systematic alteration of this ion transport pathway, measured as the rate of ouabain-sensitive ATP hydrolysis or by evaluation of the number of [³H]ouabain binding sites (for review, see Ref. 230). Several researchers reported a slight increment (by 15-25%) in the rate of ouabain-sensitive Na⁺ extrusion or K⁺ uptake in SHR erythrocytes (for review, see Ref. 304). Kuriyama and co-workers (151) demonstrated an enhanced rate of ouabain-sensitive ⁸⁶Rb⁺ uptake in SHR VSMC. In contrast, this parameter was decreased by 15% in erythrocytes from MHS (12). These results, however, were obtained under uncontrolled concentrations of intracellular Na⁺, and thus a final conclusion cannot be reached without complete kinetic study. Special attention should also be paid to the partial reaction of Na⁺-K⁺-ATPase, i.e., ouabain-sensitive Na⁺/Na⁺ and K⁺/K⁺ exchange, since they might influence the real values of net ion fluxes mediated by this ion transport system (58).

Exciting results were obtained in studies of the Na⁺-K⁺ pump in salt-sensitive hypertension. In 1993, Canessa and co-workers (28) reported that, in erythrocytes from salt-sensitive SS/JR rats, the Na⁺/K⁺ coupling ratio of 1-Na⁺-K⁺-ATPase, the sole erythrocyte isozyme, is close to 3:1 rather than 3:2, as revealed in all types of cells investigated so far, including erythrocytes from salt-resistant SR/JR rats. The same result was obtained under analysis of net ouabain-sensitive Na⁺ and K⁺ fluxes and ouabain-sensitive Na⁺/Na⁺ and K⁺/K⁺ exchange, an additional mode of operation of this transporter in salt-sensitive and salt-resistant rat strains developed in the Pavlov Institute of Physiology, St. Petersburg, Russia (155). A difference in ouabain-sensitive Na⁺ and K⁺ fluxes between erythrocytes from SS/JR and SR/JR rats was not detected by Zicha and Duhm (306). However, in this study, the K⁺/Na⁺ ratio of ouabain-sensitive fluxes was close to 1:1, which was probably caused by the presence of phosphate in the incubation medium. Indeed, it is well documented that phosphate penetrates erythrocytes through the anion exchanger and the augmented K⁺/K⁺ exchange mode of the pump (58). Putative substrain differences in Dahl rats from different colonies can also contribute to this discrepancy. Mathematical simulation showed that functional manifestation of altered modes of operation of the renal cell-specific 1/1 Na⁺-K⁺ pump in proximal tubules (3Na⁺:1K⁺) is sufficient to explain the increased reabsorption of salt and osmotically obliged water observed in salt-sensitive hypertension (210). Direct verification of this hypothesis is complicated due to methodological problems in accurate measurement of the stoichiometry of the Na⁺-K⁺ pump in nonerythroid cells.

In contrast to conflicting results on the activity of the erythrocyte Na⁺-K⁺ pump in primary hypertension, two laboratories reported that the maximal activity (V_max) of Na⁺-K⁺-ATPase is increased by 60-80% in membrane fractions from the MHS kidney cortex compared with the Milan normotensive strain (MNS) (176, 219). ATP-dependent, ouabain-sensitive Na⁺ uptake was also elevated in basolateral membrane inside-out vesicles from the kidney cortex of MHS. Because these differences were not linked to an increased number of ouabain-binding sites, they suggest altered properties of the pump itself or of its regulatory pathways (219). The possible mechanism of this abnormality is discussed in Systems Involved in Regulation of the Activity of Ion Transporters: Role of the Cytoskeleton Network.

Ca²⁺-Activated K⁺ Channels

In the mid-1980's, it was reported that outward K⁺ fluxes and membrane hyperpolarization induced by elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) are augmented in erythrocytes from SHR (98, 202, 205) but not in MHS red blood cells (202). The level of Ca²⁺-induced hyperpolarization was also increased in erythrocytes from some patients with essential hypertension (202), suggesting an enhanced activity of Ca²⁺-activated K⁺ channels. Later on, heightened conductance of Ca²⁺-activated K⁺ channels was documented by studying Ca²⁺-induced ion currents in SHR VSMC with electrophysiological methods (6, 64, 246). Increased hyperpolarization was observed in platelets and VSMC from SHR by measurement of Ca²⁺-induced shifts in membrane potential using fluorescent dyes (P. V. Avdonin, personal communication), which also suggests hyperactivity of Ca²⁺-activated K⁺ channels.

Three subclasses of Ca²⁺-activated K⁺ channels have been defined in nucleated cells based on their electrophysiological and pharmacological profiles as follows: 1) small-conductance (10-20 pS) channels that are unaffected by membrane potential and are sensitive to the bee venom toxin apamin, 2) intermediate-conductance channels (25-100 pS) that are also non-voltage gated but are insensitive to the above-mentioned drug, and 3) high-conductance (maxi-K or BK) channels (100-300 pS) that are voltage dependent and inhibited by charybdotoxin (135). The abnormalities of Ca²⁺-activated K⁺ fluxes in VSMC are probably caused by BK channels. This hypothesis was indirectly proved by measuring resting artery strip contraction. In these experiments, it was shown that application of charybdotoxin produces contraction in artery strips from SHR but not from WKY (6). The relative contribution of apamin- and charybdotoxin-sensitive K⁺ channels to enhanced Ca²⁺-activated K⁺ fluxes in erythrocytes and platelets from SHR and patients with essential hypertension has not yet been examined. It is important to mention here that voltage insensitivity and low conductance (10-20 pS) impart unique properties to erythrocyte charybdotoxin-sensitive channels (for more details, see Ref. 195). Both the BK - and -subunits involved in charybdotoxin binding have been cloned from human and mouse cDNA libraries (214). However, the genes encoding these proteins have not been mapped.

	RELATIONSHIP BETWEEN ABNORMAL ION TRANSPORT AND HYPERTENSION

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

Data presented in IDENTIFICATION OF ALTERED MONOVALENT ION TRANSPORTERS IN PRIMARY HYPERTENSION show that NHE1 is increased in SHR and in one-half of the patients with essential hypertension. Erythrocytes from MHS, SHR, and ~25% of the patients with essential hypertension possess an increased activity of bumetanide-sensitive Na⁺ and K⁺ fluxes, thus indicating an enhanced activity of NKCC1. Data from several laboratories suggest that the enhanced activity of NHE3, NKCC2, Ca²⁺-activated K⁺ channels, and amiloride-sensitive Na⁺ channels, as well as an altered mode of operation of the Na⁺-K⁺ pump, can also contribute to altered monovalent ion transport across the plasma membrane in primary hypertension. In this section, we summarize data on the relationship between abnormalities of the above-listed ion transporters and hypertension.

Abnormal Ion Transport Is Not a Consequence of Chronic Hypertension

Similar to other membrane-bound proteins, the function of ion transporters is under the control of membrane lipid composition and viscosity. Indeed, evidence exists that the plasma level of triglycerides and cholesterol affects the activity of ion transporters in erythrocytes (33, 149, 276) and platelets (307). Long-term differences in salt diet can also modify the properties of ion transporters in these cells (46, 189). Hence, it may be assumed that the altered ion transport observed in primary hypertension is not due to an intrinsic cellular property but is caused by chronic exposure to a hypertensive milieu. In other words, the abnormalities may be considered a consequence of the disease rather than a genetically determined feature of primary hypertension. However, the evidence listed below argues against this assumption.

First, in addition to freshly isolated blood cells, aortic strips, and proximal tubules, the enhanced activity of the Na⁺/H⁺ exchanger was revealed in cultured VSMC, renal epithelial cells from SHR, and immortalized lymphoblasts from patients with essential hypertension (Table 2). These cells were subjected to long-term incubation under conditions that rule out the extrinsic milieu as a causal factor of altered ion transport.

Second, in an elegant study by Bianchi and co-workers (12), (MHS × MNS) F₁ hybrids were subjected to X-ray irradiation and bone marrow transplantation from parental donor strains. Na⁺-K⁺-2Cl cotransport in erythrocytes from F₁ MHS bone marrow recipients was increased to the same extent as in donor strains compared with MNS bone marrow recipients.

Third, numerous studies performed in different laboratories revealed no systematic alteration of ion transport in blood cells from patients with renal hypertension as well as in experimental rat models of secondary hypertension (see Refs. 109, 230, 305). Fortuno and co-workers (77) reported data on a partial correction of abnormalities of erythrocyte Na⁺/H⁺ exchanger under long-term treatment with the angiotensin-converting enzyme (ACE) inhibitor quinapril. However, it is still unclear whether this effect was caused by blood pressure reduction or by direct interaction of the drug or its metabolites with cellular systems controlling the activity of the carrier. Indeed, using another ACE inhibitor, enalapril, Rosskopf and co-workers (242) found no modulation of the enhanced Na⁺/H⁺ exchanger in platelets from patients with essential hypertension.

Fourth, abnormal monovalent ion transport was demonstrated in young SHR and MHS before the development of hypertension (12, 43, 118, 231), as well as in young offspring of patients with essential hypertension (1, 270, 299).

Taken together, these data strongly suggest that abnormalities of ion transporters can be viewed as a genetically determined feature of primary hypertension rather than a consequence of long-term elevation of blood pressure.

Cosegregation of Ion Transport Abnormalities With Elevated Blood Pressure

The involvement of abnormal ion transport in the pathogenesis of primary hypertension was examined in the studies of Na⁺-K⁺-2Cl cotransport and Na⁺/H⁺ exchange in erythrocytes from F₂ hybrids of normotensive rats and SHR.

Figure 1 shows that both (MHS × MNS) F₂ hybrids (12) and (SHR × WKY) F₂ hybrids (144) possess a statistically significant positive correlation between erythrocyte Na⁺-K⁺-2Cl cotransport activity, measured as outward bumetanide-sensitive Na⁺ efflux, and blood pressure, with r = 0.520 and 0.509, respectively (P < 0.001). In addition, a study on bumetanide-sensitive ⁸⁶Rb⁺ influx in (SHR × WKY) F₂ hybrids confirmed the positive correlation of the carrier's activity with blood pressure (r = 0.301, P < 0.01) (225).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Correlation between erythrocyte Na+-K+-2Cl cotransport activity and blood pressure (BP) in F2 hybrids of Milan hypertensive strain × Milan normotensive strain (Ref. 12; A) and spontaneously hypertensive rats (SHR) × normotensive Wistar-Kyoto rats (WKY; Ref. 144; B). Values given are mean blood pressure (A) or blood pressure measured by direct methods before the animals were killed (B). Na+-K+-2Cl cotransport activity is presented as mmol · l cells1 · h1 (A) or as arbitrary units (B). Means ± SE of blood pressure and Na+-K+-2Cl cotransport activity for WKY () and SHR () progenitors are shown in B.

We did not find a significant correlation between the rate of erythrocyte µ_H⁺-induced Na⁺/H⁺ exchange and blood pressure in 35 F₂ hybrids of SHR × WKY (r = 0.196, P = 0.06) (196). However, two facts should be taken into account when analyzing these results. First, the mean values of µ_H⁺-induced Na⁺/H⁺ exchange in erythrocytes from (SHR × WKY) F₂ hybrids were two- and threefold higher than in WKY and SHR progenitors, respectively. These results can be explained as a consequence of complex gene-gene interactions. Second, as mentioned in Can We Use Erythrocyte Na⁺/Li⁺ Countertransport as a Marker of NHE1 Activity?, the properties of erythrocyte µ_H⁺-induced Na⁺/H⁺ exchange and NHE1 in nucleated cells are significantly different, and nucleated cells (listed in Table 2) rather than erythrocytes should be used for the study of cosegregation of NHE1 activity and hypertension.

Genes Encoding Ion Transporters and Quantitative Trait Loci of Hypertension

Although the consideration of data obtained in quantitative trait loci (QTL) studies may appear discouraging because of the large number of potential hypertensive genes in different loci, it may help us to conclude whether or not genes encoding ion transporters or the most potent regulators of their activity are localized in loci that cosegregate with elevated blood pressure or with cardiac hypertrophy, hyperurecemia, albuminuria, stroke latency, and other phenotypes related to damage of target organs of hypertension, such as the heart, vessels, kidney, and brain.

The search for QTL in essential hypertension meets with many problems dealing with heterogeneity in the human population, shortage of large and well-characterized pedigrees, as well as with variability of environmental factors modifying the penetrance of disease. Considering this, QTL analysis of F₂ and backcross hybrids derived from inbred SHR and their normotensive counterparts has a substantial advantage. Moreover, rat models constitute a unique tool to dissect the genetic background of primary hypertension using recombinant inbred and congenic strains. With these approaches, >30 chromosome loci were shown to be related to elevated blood pressure and other intermediate phenotypes of hypertension. Table 6 indicates that the majority of these loci exhibit strain specificity. The gender and age of animals should also be considered when analyzing these data (36, 116). Despite this diversity, three loci [1(b), 2(a), and 10(b)] possess remarkable stability in all crosses studied so far (for more details see Ref. 110).

View this table: [in this window] [in a new window]   Table 6.   QTL and intermediate phenotypes of spontaneous hypertension in rats

Recently, Bihoreau and colleagues (15) published a linkage map for all rat chromosomes. However, the density of the Rattus norvegicus genome map is still much reduced when compared with that of humans and mice. To broaden QTL analysis and enrich loci with candidate genes, we employed a comparative mapping technique. Using information on rat mapped genes collected from the "Rat Genome Database" in the Göteborg University server (http://ratmap.gen.gu.se), we retrieved data on chromosomal localization of the gene and on the human homologue. Then, using "Online Mendelian Inheritance in Man," the database created by Dr. V. A. McKusick (http://www3.ncbi.nlm.nih.gov/Omim), we proceeded to search for genes with positions close to the "anchor gene." This comparative genome mapping technique allows identification of homologous chromosomal regions in rats and humans. In most cases, construction of the synthenic map is impeded by evolutional chromosomal rearrangements that changed the gene order. For example, the order of QTL 1(b)-1(c)-1(d) in rats corresponds to at least five chromosomal segments in humans: (11p15)-(16q24-q13)-(3p)-(16p11-p13)-(19q13-q12). However, for some regions, homology was proved for the long piece of the chromosome. Thus locus 5(a)-5(b) corresponds to the human chromosomal region 1p34-pter, and QTL 10(a)-10(b) matches 17p13-17q24.

The comparative genome mapping approach is useful to establish a precise localization of the gene, even if it is not yet mapped. Thus chromosomal homology for rat chromosome 5 and human chromosomal piece 1p was reported by Szpirer and co-workers (280). QTL 5(a) and 5(b) were mapped around Anf and Et2 genes (Table 6). Using these genes as anchors and information on homologous human genes, we constructed a synthenic map for this region and were able to localize the NHE1 gene more precisely within locus 5(b) (Table 6). This region of chromosome 5 contains QTL for elevated blood pressure, stroke latency, and sensitivity to cerebral ischemic insult in F₂ SHR × WKY and SHRSP × WKY (132, 243, 303). Data obtained for other regions with this analysis are briefly summarized below.

First, genes encoding the - and -subunits of ENaC, the 3-, 1-, and 2-subunits of the Na⁺-K⁺ pump, and NKCC2 are localized within QTL 1(b), 1(d), 3(c), and 10(a). These loci are involved in elevated blood pressure as well as in enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency.

Second, several additional genes encoding monovalent ion transporters can be predicted within QTL under synthenic analysis of human and rat genome maps in accordance with the methodology described above: 1(b), NCC and NHE5; 2(a), 2-subunits of the Na⁺-K⁺ pump; 4(d), -ENaC; 5(b), -ENaC and NHE1; 18(b), NKCC1.

Third, several lines of evidence suggest that, apart from monovalent ion transporters, hyperactive L-type Ca²⁺ channels and the plasma membrane Ca²⁺ pump contribute to intracellular Ca²⁺ overloading in primary hypertension (for recent review, see Ref. 194). Table 6 shows that QTL 1(b), 3(b), 4(d), 10(a), 13, and X contain mapped or provisionally positioned genes encoding different subunits of L-type Ca²⁺ channels (Cacn) and pump (Atp2).

Fourth, data presented in IDENTIFICATION OF ALTERED MONOVALENT ION TRANSPORTERS IN PRIMARY HYPERTENSION indicate an enhanced activity of renal cell-specific isoforms of the Na⁺/H⁺ exchanger (NHE3) and charybdotoxin-sensitive Ca²⁺-activated K⁺ channels (BK). QTL listed in Table 6 do not contain NHE3. Genes encoding BK subunits were not yet mapped.

An analysis of other genes inside QTL that could be related to the pathogenesis of hypertension was performed recently (110).

	POSSIBLE MECHANISMS INVOLVING ABNORMAL ION TRANSPORTERS IN THE PATHOGENESIS OF HYPERTENSION

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

Data presented in RELATIONSHIP BETWEEN ABNORMAL ION TRANSPORT AND HYPERTENSION show that abnormalities of ion transport pathways seen in several types of cells from SHR and patients with essential hypertension are not caused by the hypertensive milieu and may be involved in the pathogenesis of this disease as a causal factor or as a factor of susceptibility for environmental stress and the development of complications in target organs of hypertension. Despite the widespread characteristics of ion transport abnormalities, their manifestations in a limited number of cell types point to their influence on the pathogenesis of the disease. Indeed, complete substitution of bone marrow in (MHS × MNS) F₁ hybrids with bone marrow from progenitors changed the ion transport properties of mature erythrocytes toward those of donor strains but did not affect blood pressure (12), demonstrating that altered ion transport in blood cells per se is not involved in the pathogenesis of hypertension. This section deals with the possible mechanism of ion transport abnormalities in the pathogenesis of primary hypertension via their manifestation in the kidney epithelium and vascular smooth muscle (Fig. 2). This approach is based on the well-documented major role of kidney resetting and enhanced peripheral vascular resistance in long-term maintenance of elevated blood pressure (99).

View larger version (64K): [in this window] [in a new window]   Fig. 2.   Possible mechanisms of involvement of altered monovalent ion transporters in pathogenesis of primary hypertension via modification of vascular smooth muscle cell (VSMC) and renal epithelial cell (REC) function. BP, blood pressure; DLS, digitalis-like substances; EFV, extracellular fluid volume; ER, endoplasmic reticulum; W/L, wall-to-lumen vessel ratio. See text for more details.

Ion Transport Abnormalities and Kidney Function

The key role of the kidney in long-term blood pressure elevation was originally proposed by Guyton et al. (100) and proven by transfer of blood pressure differences between MHS and MNS (13) as well as Dahl salt-sensitive and salt-resistant rats (44) after kidney transplantation. The same results were obtained in long-term studies on the effect of kidney transplantation from normotensive donors, with or without a family history of hypertension, on the development of hypertension in recipients (94). The pressure effect of transplantation seems to associate with the different activity of ion transporters, since the kidney from MHS and SHR caused accelerated Na⁺ reabsorption across the tubular epithelium (for more details see Ref. 72). A major role of kidney ion transporters in long-term blood pressure regulation was also supported by recent data on the identification of mutation sites in monogenic forms of hypertension and hypotension (see Search for mutations).

Several lines of evidence presented in Na⁺/H⁺Exchange, Cl-Dependent Cotransporters, and Amiloride-Sensitive Na⁺ Channels suggest that increased reabsorption of salt and osmotically obliged water in low-renin hypertension can be caused by enhanced activity of NHE3, ENaC, and NKCC2, which are mainly localized in the brush-border membrane of the proximal tubule and thick ascending limb of Henle's loop. An altered mode of operation of the 1-Na⁺-K⁺ pump (see Na⁺-K⁺ Pump) can also contribute to this phenomenon (Fig. 2). It was shown that transgenic mice overexpressing NHE1 exhibit decreased urinary excretion and elevated systolic blood pressure after excessive salt intake (153). Recent data on the antihypertensive effect of intracerebroventricular infusion of amiloride into rats with DOCA-salt hypertension (184) suggest that, besides renal -ENaC, -ENaC can also be involved in the development of Na⁺-induced hypertension. However, the mechanism by which basolateral NHE1 and nonepithelial -ENaC regulate kidney function is not currently well understood.

Ion Transport Abnormalities and Vascular Smooth Muscle Function

Vascular tone. The hypothesis of the involvement of monovalent ion transporters in the regulation of vascular tone was initially based on cross talk of the Na⁺/H⁺ exchanger and Na⁺/Ca²⁺ exchanger in the regulation of [Ca²⁺]_i (17). In its initial form, this hypothesis was criticized due to the lack of systematic indications of enhanced free intracellular Na⁺ concentration ([Na⁺]_i) in VSMC of hypertensive animals and relatively low activity of the VSMC Na⁺/Ca²⁺ exchanger compared with other electrical excitable tissues (228, 230). However, the hypothesis was revised recently after analysis of intramembrane compartmentalization of the above-mentioned ion transporters. An immunohistochemical approach showed that the 3-subunits of the Na⁺-K⁺ pump possessing the highest affinity for ouabain and for endogenous digitalis-like substances (DLS) (51, 112) as well as the Na⁺/Ca²⁺ exchanger are colocalized in the same region of the VSMC sarcolemma overlying the endoplasmic reticulum, whereas the 1-subunits of the Na⁺-K⁺ pump and the Ca²⁺ pump are diffusely distributed along the sarcolemma. The compartment, abundant in 3-Na⁺-K⁺ pump and Na⁺/Ca²⁺ exchanger, was called plasmerosome (90, 134). On the basis of these results, it was proposed that [Na⁺]_i and [Ca²⁺]_i in plasmerosomes differ from those in the bulk of the cytoplasm, and abnormalities of intracellular Ca²⁺ handling via enhanced [Na⁺]_i, intracellular Na⁺/extracellular Ca²⁺ exchange, and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum are limited only to the plasmerosome region (134). Enhanced activity of Ca²⁺-activated K⁺ channels (see Ca²⁺-Activated K⁺ Channels) can be viewed as a feedback mechanism that partly protects VSMC from hypercontractility.

The concept of compartmentalization of Ca²⁺ and Na⁺ transporters in VSMC suggests cross talk between renal and vascular mechanisms in the development of hypertension. Indeed, expansion of extracellular volume caused by enhanced reabsorption of salt and water in the renal epithelium leads to increased production of DLS (51, 112). These compounds try to normalize extracellular fluid volume via inhibition of the Na⁺-K⁺ pump in the tubular epithelium. However, the affinity for DLS of the 1-subunit of the Na⁺-K⁺ pump in these cells is lower than that of the 2- and 3-subunits expressed in VSMC and sympathetic nerve terminals (70, 281). By acting predominantly on these cells, DLS increase [Ca²⁺]_i via activation of the intracellular Na⁺/extracellular Ca²⁺ exchanger. Moreover, because of the electrogenicity of the Na⁺-K⁺ pump, DLS hyperproduction would lead to membrane depolarization and opening of L-type Ca²⁺ channels (see Fig. 2).

Vascular remodeling. The altered geometry of vessels (increased wall-to-lumen ratio) is one of the potent mechanisms of long-term maintenance of elevated blood pressure (76, 99). In the late 1980's, several laboratories confirmed an initial observation by Yamori and co-workers (300) of increased growth and DNA synthesis in VSMC from spontaneously hypertensive rodents under culture conditions (23, 102, 105, 212, 255). It was also established that accelerated cell growth of SHR VSMC is mostly due to a shortening of ~4 h in the G₁ phase and early entry of cells into the S phase (103). These results suggested that hypertrophy of the vessel wall in primary hypertension is caused by modified activity of gene products involved in controlling cell cycle progression rather than being a consequence of altered humoral systems and enhanced hemodynamic pressure.

Several lines of evidence point to cross talk between the activity of ion transporters, cell volume regulation, and cell cycle progression. First, it was shown that enhanced activity of the Na⁺/H⁺ exchanger correlates positively with an enhanced rate of proliferation in VSMC from SHR (11, 158), immortalized lymphoblasts from patients with essential hypertension (183), and skin fibroblasts from IDDM patients with nephropathy (169, 285). Second, in several types of cells including VSMC, growth factors stimulate Na⁺/H⁺ exchange and Na⁺-K⁺-2Cl cotransport activities (for review see Refs. 92, 213, 296). Third, mutant fibroblasts lacking the Na⁺/H⁺ exchanger or Na⁺-K⁺-2Cl cotransporter are less responsive to growth factors (for review see Refs. 92, 156, 213). Fourth, in BALB/c 3T3 fibroblasts, addition of amiloride or bumetanide partly inhibited cell proliferation, whereas simultaneous addition of both compounds completely blocked cell cycle progression (217). In VSMC, serum-induced [³H]thymidine incorporation was completely blocked by 5 µM EIPA, a potent inhibitor of NHE1 (108). Overall, these data suggest that the increased Na⁺/H⁺ exchange or Na⁺-K⁺-2Cl cotransport activities found in primary hypertension (Tables 2 and 5) can contribute to accelerated cell cycle progression and enhanced VSMC growth. In hepatocytes, ion transporters affect proliferation via modulation of cell volume (156). The role of cell volume in the regulation of cell cycle progression in VSMC has not yet been explored.

Both VSMC growth and death contribute differently to hypertrophy, hyperplasia, and remodeling of the vessel wall (104). Enhanced occurrence of programmed cell death (apoptosis) in cultured VSMC (111, 190) and prolonged apoptotic windows in target organs of hypertension during establishment of hypertension (108) have been demonstrated in SHR. Recent studies revealed that, besides cell proliferation, ion transporters can be involved in triggering and progression of apoptosis (124). In thymic lymphocytes, hyperosmotic conditions led to the same complete apoptosis observed under dexamethasone application (18). Like the majority of cells studied so far, VSMC undergoing apoptosis show a transient cell volume decrease (190). Moreover, hyperosmotic shrinkage of VSMC potentiates apoptosis (190). However, in contrast to thymic lymphocytes lacking regulatory volume increase, volume recovery of VSMC after isosmotic shrinkage is mediated by Na⁺-K⁺-2Cl cotransport (207), thus providing a feedback mechanism for protection against factors leading to a massive loss of intracellular osmolytes. Further studies should clarify the role of abnormal ion transport pathways in the augmented progression of apoptosis in SHR VSMC and its contribution to vascular remodeling.

	MOLECULAR DETERMINANTS OF ABNORMAL ION TRANSPORT IN HYPERTENSION

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

Accelerated monovalent ion transport in primary hypertension can be caused by mutations of genes encoding ion transporters, leading to overexpression of gene products or to increased turnover number of ion carriers and open probability of ion channels. Functional abnormalities can also be triggered by the altered activity of systems involved in regulating the expression or activity of these transporters. In this section, we analyze the data concerning this topic.

Genes Encoding Ion Transporters

Search for mutations. NA⁺/H⁺ EXCHANGERS. As discussed in Na⁺/H⁺ Exchange, enhanced activity of NHE1 is one of the most potent hallmarks of altered ion transport in SHR and in patients with essential hypertension. By analysis of a single polymorphism located in the first intron of NHE1 and two polymorphisms in neighboring chromosome regions in Utah pedigrees, Lifton and co-workers (165) excluded NHE1 as a candidate gene involved in the augmented activity of Na⁺/Li⁺ countertransport as well as in essential hypertension selected by criteria of enhanced activity of this transporter. This conclusion is in line with cell physiology data on the different functional properties of NHE1 and erythrocyte Na⁺/Li⁺ countertransport (see Can We Use Erythrocyte Na⁺/Li⁺ Countertransport as a Marker of NHE1 Activity?). It should be stressed, however, that this population study did not rule out the presence of mutations in coding or untranslated regions of NHE1, which could be involved in the development of complica-tions of hypertension or even in the pathogenesis of this disease in patients with normal Na⁺/Li⁺ countertransport activity. The first hypothesis is supported by data on the presence of NHE1 within QTL of salt-induced increments of blood pressure and stroke latency (Table 6). To examine the presence of mutations in NHE1, we undertook single-stranded conformation polymorphism analysis of 23 fragments and partial sequencing of NHE1 cDNA from VSMC of stroke-prone SHR (SHRSP) possessing, similar to SHR, enhanced activity of the Na⁺/H⁺ exchanger (197). This analysis did not reveal any mutation in the coding region of NHE1 cDNA obtained by reverse transcription of mRNA from VSMC of SHR and SHRSP (188). As with NHE1, no mutation has been found in cDNA encoding kidney-specific NHE3 in SHR (118).

CL-DEPENDENT COTRANSPORTERS. Monogenic or so-called Mendelian forms of hypotension, such as pseudohypoaldosteronism type 1 (PHA-1), Gitelman's syndrome, and Bartter's syndrome, possess increased plasma renin activity and salt wasting. Hyperkalemia and metabolic acidosis are additional markers of PHA-1, whereas hypokalemia, decreased Ca²⁺ extrusion, and plasma Mg²⁺ content are obligatory components of the pathogenesis of Gitelman's syndrome. In Bartter's syndrome, hypokalemia is accompanied by metabolic acidosis and hyperaldosteronism. About 20 mutations leading to inactivation of NCC have been detected in patients with Gitelman's syndrome up to now (267, 268). Bartter's syndrome is linked to D648N and V272F mutations in NKCC2, causing inactivation of this carrier in affected patients (266, 267). To the best of our knowledge, no analysis has been conducted of cDNA encoding Cl-dependent cotransporters in primary hypertension.

ENAC. Liddle's syndrome is the most studied form of monogenic hypertension. This disease is characterized by hypertension that responds well to low-salt diet combined with treatment by amiloride or other ENaC inhibitors. This syndrome is associated with salt retention, hypokalemia, and suppressed aldosterone secretion and plasma renin activity. Liddle's syndrome in Caucasian and Japanese families is linked to a segment of chromosome 16 that contains two candidate genes encoding the - and -subunits of ENaC (113, 259). Studies of these genes in patients with Liddle's syndrome and their products in transfected cells demonstrated a set of mutations leading to deletion of the cytoplasmic COOH terminus and constitutive activation of ENaC (for more details, see Refs. 164 and 258). Genetic analysis of affected offspring revealed a linkage of PHA-1 with loci on chromosomes 12 and 16 that contain genes encoding the - and -subunits of ENaC. However, in contrast to Liddle's syndrome, in this form of monogenic hypotension, mutations are located in the extracellular loop of the -subunit or in the NH₂ terminus of the - and -subunits, leading to suppression of ENaC activity.

In a reciprocal cross between SHRSP and WKY, gene loci containing ENaC - and -subunits cosegregate with salt-induced increments in blood pressure. However, comparative analysis of the complete coding sequences of the -, -, and -subunits in SHRSP and WKY revealed no biologically relevant mutations (148). Negative results were also obtained in the study of -ENaC in Japanese patients with essential hypertension (32).

Low-renin hypertension is more common in blacks than in whites and, like patients with Liddle syndrome, blacks possess a low urine aldosterone-to-K⁺ ratio. Persu and co-workers (223) reported that the frequency of genetic variants of -ENaC in French Caucasians with essential hypertension is <1% but reaches 44% in French of African origin. Seven mutations located at exons 8 (V434M, G442V) and 12 (G589S, T594M, R597H, R624C, E632G) contribute to the genetic variants in blacks with essential hypertension. To analyze the functional consequence of these mutations, wild-type and mutated -ENaC were coexpressed with normal - and -ENaC in Xenopus oocytes. This study demonstrated that both amiloride-sensitive current and ²²Na⁺ uptake are increased by 30-50% in oocytes transfected with G589S-mutated -ENaC, whereas the remaining mutations did not alter the activity of amiloride-sensitive Na⁺ channels measured under basal conditions. Clinical evaluation of a family bearing the G589S variant did not reveal cosegregation of this mutation with hypertension (223).

Under analysis of the functional consequence of other mutations of -ENaC in blacks, it was noted that T594M and R624C mutations abolish putative protein kinase C phosphorylation sites. In addition, the T594M mutation blocks the inhibition of ENaC by activators of this enzyme (39) and leads to an enhanced effect of cAMP on ENaC activity in lymphocytes from patients bearing this mutation (278). A similar frequency of the T594M mutation was reported in normotensive and hypertensive African Americans (278). In contrast, the analysis of 438 black residents of London by Baker and co-workers (7) revealed that the T594M mutation is more frequent in patients with essential hypertension compared with their normotensive counterparts (8.3 and 2.1%, respectively).

Apart from mutations of the -subunit, an A344T mutation of -ENaC (with frequencies of 0.426 and 0.054 in blacks and whites, respectively) has been reported (233). However, the relevance of this finding to ENaC function and to essential hypertension in blacks remains unclear. There are no data on the analysis of mutations of the nonepithelial -ENaC subunit in primary hypertension.

NA⁺-K⁺ PUMP. Adenine-1079 to thymidine transversion in the coding region of the 1-Na⁺-K⁺ pump gene leading to G276L substitution was identified by sequencing kidney cDNA libraries from salt-sensitive SS/JR rats (120). The restriction fragment length polymorphism performed in this study showed that SS/JR and SR/JR rats are homozygous for the thymidine-1079 and adenine-1079 alleles, respectively. Simonet et al. (269) failed to reproduce these data using restriction enzyme analysis of 1-Na⁺-K⁺ pump cDNA fragments amplified with Taq polymerase by PCR. Subsequently, Ruiz-Opazo and co-workers (244) took a Taq polymerase-independent approach based on polymerase allele-specific amplification and ligase chain reaction analysis of kidney mRNA subjected to RT-PCR. This study showed that the negative result obtained by Kurtz and co-workers was probably caused by a consistent Taq polymerase chain reaction error that selectively substituted adenine-1079 for thymidine. With the use of the Xenopus laevis oocyte expression system, it was demonstrated that cells carrying total kidney RNA or cDNA derived from in vitro transcribed 1-mRNA of SS/JR rats exhibit decreased ouabain-sensitive ⁸⁶Rb⁺ influx compared with SR/JR rats (120). However, the relationship of this finding to altered 1-Na⁺-K⁺ pump activity or to the Na⁺/K⁺ coupling ratio in salt-sensitive hypertension (see Na⁺-K⁺ Pump) is still unknown.

Expression of genes encoding ion transporters. This issue was studied in more detail with the Na⁺/H⁺ exchanger. Indeed, based on the data presented in Table 3, it may be suggested that the increased V_max of NHE1 in primary hypertension is caused by overexpression of the carrier due to a mutation in the noncoding region of NHE1 or to altered activity of intracellular pathways controlling the expression of this carrier. Table 7 shows that Northern blot analysis did not reveal overexpression of NHE1 mRNA in VSMC and proximal tubules from SHR. In addition, no differences were observed in NHE1 protein content in VSMC and proximal tubules between SHR and WKY by two groups of researchers. LaPointe and co-workers (157) reported that NHE1 protein content is increased in VSMC from SHR between the 2nd and 10th passages but is not altered in freshly isolated aorta from SHR. When analyzing the latter results, it is important to recall that both cultured VSMC and VSMC in freshly isolated aorta exhibit enhanced Na⁺/H⁺ exchanger activity (Table 2). Garciandia and co-workers (82) reported that NHE1 mRNA content is increased by 30% in lymphocytes from patients with essential hypertension. This result contradicts data on unaltered NHE1 mRNA and protein expression in lymphoblasts from essential hypertensive subjects (183, 241) and on unaltered expression of NHE1 mRNA in subgroups of essential hypertensive patients with high and normal lymphocyte Na⁺/H⁺ exchange activity (77). While Northern blot analysis did not reveal changes in NHE3 mRNA content in proximal tubules from SHR (118), NHE3 protein was found to be increased by 50% in SHR compared with WKY (140). Taken together, these data suggest that the transcription of NHE1/NHE3 is not altered in primary hypertension. Additional studies should be performed to further examine this hypothesis as well as to examine the efficiency of NHE1/NHE3 translation or degradation as a possible mechanism of enhanced Na⁺/H⁺ exchanger activity in primary hypertension.

View this table: [in this window] [in a new window]   Table 7.   Expression of mRNA and protein level of NHE1 and NHE3 in primary hypertension

Comparison of mRNA content encoding -, -, and -subunits of ENaC in kidney showed no differences between SHRSP and WKY (148). Negative results were also obtained when comparing NKCC2 mRNA expression in kidneys from SHRSP and WKY (173). Up to now, there is no indication of altered expression of NKKC1 as well as of - and -subunits of the Na⁺-K⁺ pump in primary hypertension. It should be stressed that interpretation of the data on expression of the Na⁺-K⁺ pump in salt-loaded rats is complicated because of the presence of Na⁺-sensitive elements in the promoter region of genes encoding its 1-, 2-, and 1-subunits (245).

Systems Involved in Regulation of the Activity of Ion Transporters: Role of the Cytoskeleton Network

The data summarized in Genes Encoding Ion Transporters show that evidence for mutations in genes encoding ion transporters in primary hypertension is limited to the observation of G276L substitution in 1-Na⁺-K⁺ pump subunits from Dahl salt-sensitive rats and more frequent T594M mutation of the -subunit of ENaC in the black population with essential hypertension. No mutations have been revealed in the coding regions of NHE1 and NHE3, which exhibit enhanced activity in SHR. These results suggest that alteration of ion transporters in primary hypertension is caused by abnormalities of the systems involved in regulation of their expression in the plasma membrane or involved in functional activity, rather than by direct mutation in the coding portion of their genes. Use of the candidate approach to search for causal genes is extremely difficult due to the large diversity of intracellular signaling pathways related to the regulation of monovalent ion transport. Thus, for example, NHE1 activity is under the control of receptor tyrosine kinases and seven membrane-spanning receptors coupled to GTP-binding proteins, mitogen-activated protein kinase kinases, phospholipases, Ca²⁺-regulated enzymes, and cell volume (for more details, see Ref. 296). This issue becomes even more complicated due to the tissue-specific character of these regulatory pathways. Indeed, in different cell types, an activator of protein kinase A can either inhibit, activate, or not affect NKCC1. The same results were obtained for activators of protein kinase G and protein kinase C (79). Analysis of the activity of signaling systems in primary hypertension and their cross talk with monovalent ion transporters is outside the scope of the present review. Several aspects of this topic are covered by other reviews (109, 284).

It should be stressed, however, that the candidate gene approach based on our current knowledge of intracellular signaling mechanisms can be misleading in the search for biochemical determinants of altered ion transport in primary hypertension because of the presence of a set of additional unidentified regulatory pathways. Thus, for example, Table 3 shows that altered NHE1 activity in primary hypertension is caused by enhanced V_max rather than by altered affinity of the carrier for intracellular H⁺. On the contrary, as is shown in Fig. 3A, hormones and neurotransmitters that raise [Ca²⁺]_i, as well as growth factors and activators of protein kinase C (92, 185, 296), augment Na⁺/H⁺ exchanger activity by increasing its sensitivity to intracellular H⁺, without affecting the maximal activity of the carrier. Considering this, the involvement of these regulators in the elevation of NHE1 in primary hypertension should probably be ruled out. Unlike the above-mentioned modulators of NHE1, cell volume (Fig. 3B), NaF, a nonspecific activator of GTP-binding proteins (188), and the newly discovered G₁₃ and G₁₂ (Fig. 3C) affect the V_max of the Na⁺/H⁺ exchanger rather than its affinity for intracellular H⁺. Intracellular signaling can also be mediated by the complex of activated GTP-binding proteins (292). Furthermore, recently, Siffert and co-workers (265) detected C825T polymorphism leading to deletion of 41 amino acids in the 3-subunit of GTP-binding proteins. Genotype analysis of 853 subjects suggested a significant association of the mutated allele with essential hypertension.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Dependence of Na+/H+ exchange activity on intracellular pH under control conditions (line 1 in A, B, and C), Ca2+-ionophore-induced increment of intracellular free Ca2+ concentration (line 2 in A; Ref. 295), cell shrinkage (line 2 in B; Ref. 131), and addition of mutationally activated -subunits of GTP-binding proteins [line 2 (G13) and line 3 (G12) in C; Ref. 166]. Maximal activity of Na+/H+ exchanger in control (untreated) cells was taken as 100%.

Side by side with the possible implication of GTP-binding proteins, several observations listed below indicate that abnormalities of cytoskeleton organization are involved in the altered activity of ion transporters in primary hypertension. First, in VSMC (208) and in renal epithelial cells (174, 175), modulation of Na⁺-K⁺-2Cl cotransport by activators of cAMP signaling was accompanied by cytoskeleton reorganization, and this was mimicked or blocked by compounds affecting different components of the cytoskeleton network. Second, in erythrocytes and renal epithelial cells, thermally induced rearrangement of the cytoskeleton network is sufficient to induce a drastic modulation of the basal activity of several ion transporters, including the Na⁺-K⁺-2Cl cotransporter and Na⁺/H⁺ exchanger, and to abolish their hormonal and volume-dependent regulation (78, 191). Third, G₁₃-induced activation of NHE1 was accompanied by stress fiber formation in fibroblasts (123), suggesting that, like the volume-dependent regulation of ion transporters (156, 192), this signaling pathway is also mediated by cytoskeleton reorganization. Fourth, in contrast to other ion transporters covered by this review, cellular mechanisms controlling the activity of Na⁺/Li⁺ countertransport are poorly understood (see Can We Use Erythrocyte Na⁺/Li⁺ Countertransport as a Marker of NHE1 Activity?). Recently, it was reported that the activity of this carrier may be altered by two- to threefold under modification of a minor component of the erythrocyte cytoskeleton, a newly discovered 33-kDa protein (247). Fifth, 100-fold dilution of cytoplasmic constituents during preparation of resealed ghosts did not abolish the difference in activity of the Na⁺-K⁺-2Cl cotransporter between MHS and MNS erythrocytes (74), showing that NKCC1 activation in primary hypertension is not caused by cytoplasmic components of signaling pathways. In contrast, disruption of the erythrocyte cytoskeleton under preparation of inside-out vesicles drastically affected the properties of furosemide/bumetanide-sensitive Na⁺ and K⁺ fluxes (97) and completely abolished the differences between MHS and MNS (73).

The first direct evidence of abnormal cytoskeleton organization in primary hypertension was probably provided by the observation of an altered profile of heat absorption by cytoskeleton proteins, revealed by scanning microcalorimetry in intact and cytoskeleton-depleted erythrocyte membranes from SHR (95) and by electron paramagnetic resonance studies of membrane-bound protein mobility in erythrocytes from SHR (96). In erythrocytes, the cytoskeleton network is formed by heterodimers of - and -spectrin and actin bundles. This network is assembled by means of a set of anchor proteins that includes adducin (10, 171), a heterodimeric / protein that plays a major role in the assembly of actin-based cytoskeleton through precise regulation of actin filament length. Table 6 shows that genes encoding adducin , , and  are located in chromosome fragments 1b, 4c, and 14, which were identified as QTL for elevated blood pressure and its complications. A few years ago, an immunochemical difference of erythrocyte adducin between MHS and MNS led to the subsequent identification of missense mutations in cDNA encoding  (F316Y),  (Q529R), and  (Q572K) adducin in MHS (14, 287). Furthermore, -adducin polymorphism cosegregates with blood pressure in F₂ MHS × MNS hybrids (14). There is no cosegregation of - and -adducin polymorphism per se with blood pressure, but the MHS-like allele of these genes potentiates the segregation of blood pressure with -adducin polymorphism (14, 287). For our discussion, it is important to say that, besides modulation of the cytoskeleton assembly, mutated adducin from MHS leads to a 40% increase in Na⁺-K⁺ pump V_max in transfected epithelial cells (288). The relationship of this mutation to regulation of the activity of other ion transporters has not yet been examined.

The presence of mutation in human -adducin leading to G460W substitution was revealed in a population study of Caucasians from Italy with a frequency distribution of GG, GW, and WW alleles of 60, 37, and 3%, respectively. Furthermore, it was shown that, in patients with GW and WW alleles, the probability of salt-induced hypertension was eightfold higher than in patients with the WW allele (40). These data constitute the first example of the association of polymorphism occurring in the same gene with hypertension in rats and humans. It should be mentioned, however, that, in contrast to hypertensive subjects from Italy, no association between hypertension and -adducin G460W polymorphism was established in studies of hypertensive and normotensive subjects from Japan (139) and Scotland (136). These observations and data on the T594M -ENaC mutation limited to black hypertensive subjects from London (see Search for mutations) as well as the inconstancy between populations for a number of other proposed genes, such as ACE, angiotensinogen, and the Sa gene, underlie the complex race- and population-dependent interplay of gene-gene and gene-environment interactions in the maintenance of elevated blood pressure in essential hypertension (110, 115).

	CONCLUSION AND PERSPECTIVES

Top Abstract Introduction IDENTIFICATION OF ALTERED... RELATIONSHIP BETWEEN ABNORMAL... POSSIBLE MECHANISMS INVOLVING... MOLECULAR DETERMINANTS OF... CONCLUSION AND PERSPECTIVES References

The data summarized in IDENTIFICATION OF ALTERED MONOVALENT ION TRANSPORTERS IN PRIMARY HYPERTENSION show that enhanced activity of the Na⁺/H⁺ exchanger, Na⁺-K⁺-2Cl cotransporter, epithelial Na⁺ channels, and Ca²⁺-activated K⁺ channels and the altered mode of operation of the Na⁺-K⁺ pump contribute to altered membrane permeability of monovalent ions in primary hypertension. Several lines of evidence presented in RELATIONSHIP BETWEEN ABNORMAL ION TRANSPORT AND HYPERTENSION and POSSIBLE MECHANISMS INVOLVING ABNORMAL ION TRANSPORTERS IN THE PATHOGENESIS OF HYPERTENSION suggest that abnormalities of these ion transporters can be involved in the pathogenesis of this disease. However, the mechanisms of these alterations remain unclear. Indeed, despite identification of the genes encoding some of these ion transporters within QTL related to the pathogenesis of hypertension, analysis of cDNA structure did not reveal any mutation in the coding region of genes encoding the ubiquitous and renal cell-specific isoforms of the Na⁺/H⁺ exchanger as well as of genes encoding subunits of epithelial Na⁺ channels, with the exception of the T594M mutation of -ENaC in a limited population of blacks with essential hypertension. The involvement of single point mutations of the Na⁺-K⁺ pump 1-subunit and the altered operational mode of this transporter in Dahl salt-sensitive rats have not yet been examined. Viewed collectively, these results suggest that, in contrast to Mendelian forms of symptomatic hypertension, the enhanced activity of ion transporters in primary hypertension is mainly caused by abnormalities of systems involved in the regulation of their expression and/or functioning. Keeping in mind the diversity of gene products involved in the regulation of expression and function of ion transporters, we believe that the mapping of ion transport phenotypes in F₂ hybrids and recombinant inbred strains obtained by a cross of hypertensive and normotensive progenitors as well as human genetic studies, such as mapping of QTL for altered ion transporters in affected sibling pairs, will lead to the identification of genetic mechanisms underlying these abnormalities. This positional cloning approach as well as genetic loss-of-function and gain-of-function experiments is required in studies with transgenic animals to analyze the involvement of abnormal ion transporters in the long-term maintenance of elevated blood pressure and in the complications of chronic hypertension. Besides this issue, identification of the genetic basis of ion transport abnormalities may lead to the development of individual therapies for this disease based on pharmacogenetic approaches. Thus, for example, it was shown that polymorphism in QTL 2(a) lacking genes encoding any subunits of L-type Ca²⁺ channels (Table 6) influences the blood pressure response to Ca²⁺ channel blockers (294), whereas patients bearing the G460M mutation in -adducin benefit from treatment with diuretics (40).

	ACKNOWLEDGEMENTS

We acknowledge the late Mitzy Canessa for her highly significant contribution to these studies.

	FOOTNOTES

The secretarial help of Monique Poirie and Josee Bedard-Baker and editorial assistance of Ovid Da Silva are greatly appreciated.

This work was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Canada, Pfizer Canada, Bayer Canada, and the National Institutes of Health of the United States (SCOR Program).

S. N. Orlov was a fellow of the International Society of Hypertension (Pfizer Award) and a scholar of Servier Canada. V. A. Adarichev was the recipient of a fellowship from the Medical Research Council of Canada.

Present address of V. A. Adarichev: Dept. of Pharmacology, Univ. of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612.

Address for reprint requests: S. N. Orlov, Centre de Recherche, Centre Hospitalier Universitaire Montreal, 3850 rue St-Urbain, Montreal, PQ, Canada H2W 1T8 (E-mail: orlovs{at}ere.umontreal.ca).

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