vascular sodium pump: endothelial modulation and alterations in some pathological processes and...

Pharmacology & Therapeutics 84 (1999) 249–271

0163-7258/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.PII:

S0163-7258(99)00037-6

Vascular sodium pump: endothelial modulation and alterations in some pathological processes and aging

Jesús Marín

a,b,*

, Juliana Redondo

a

a

Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma, C/ Arzobispo Morcillo 4, 28029 Madrid, Spain

b

Servicio de Farmacología Clínica, Clínica Puerta de Hierro, Madrid, Spain

Abstract

The vascular Na

1

pump maintains intracellular ionic concentration and controls membrane potential. Its inhibition by cardiac glycosides

enhances the intracellular Na

1

concentration. This in turn activates the Na

1

-Ca

2

1

exchange mechanism, which induces intracellular Ca

2

1

in-crease, membrane depolarization, and noradrenaline release from perivascular adrenergic nerve endings; mechanisms that promote vaso-constriction. This article reviews the relevance of the Na

1

pump in vascular tone regulation and the modulation of its activity by the endothe-lium. The endothelium negatively modulates the vasoconstriction elicited by Na

1

pump inhibition by the release of nitric oxide, according tosome authors, or an unknown factor, as suggested by others. The possible existence of endogenous digitalis-like factors is also reviewed, as isthe involvement of the vascular Na

1

pump in some cardiovascular disorders and aging. © 1999 Elsevier Science Inc. All rights reserved.

Keywords:

Vascular sodium pump; Hypertension; Diabetes; Aging; Ouabain-like factors; Endothelium

Abbreviations:

cPLA

2

, cytosolic phospholipase A

2

; DAG, diacylglycerol; DOCA, deoxycorticosterone acetate; EDHF, endothelium-derived hyperpolariz-ing factor; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; NO, nitric oxide; PG, prostaglandin; PKC, protein kinase C; SHR, spontaneous hypertensive

rat; VOCC, voltage-operated Ca

2

1

channel; WKY, Wistar-Kyoto.

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2502.

Structure and isoforms of the Na

1

pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2513. Na

1

pump and vascular tone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2534. Methods to characterize the vascular Na

1

pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2544.1. K

1

-induced relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2554.2. Measurement of

32

P release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2554.3. Ouabain-sensitive

86

Rb

1

uptake. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2554.4. [

3

H]Ouabain-binding experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2555. Na

1

pump and endothelial modulation on noradrenaline release. . . . . . . . . . . . . . . . . . . . . 2566. Na

1

pump and vasomotor responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2566.1. Na

1

pump and endothelium-dependent relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . 2566.2. Na

1

pump and endothelium-independent relaxation . . . . . . . . . . . . . . . . . . . . . . . . . 2566.3. Influence of the Na

1

pump on agonist-induced contractions . . . . . . . . . . . . . . . . . . 2577. Endothelial modulation of the vascular Na

1

pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2577.1. Arterial segments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2577.2. Cultured vascular smooth muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

8. Cardiac glycosides and nitric oxide production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2589. Physiological role of endogenous digitalis-like factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

10. Na

1

pump and hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26011. Diabetes and Na

1

pump activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26212. Changes in the Na

1

pump elicited by aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26213. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

* Corresponding author. Tel.:

1

34-91-397 5411; fax:

1

34-91-397 5302.

E-mail address

: [email protected] (J. Marín)

250

J. Marín, J. Redondo / Pharmacology & Therapeutics 84 (1999) 249–271

1. Introduction

The Na

1

/K

1

-ATPase or Na

1

pump (EC 3.6.37) ispresent in the plasma membrane of practically all eukary-otic cells, including vascular smooth muscle cells. Its func-tion is to maintain the transmembranous ion balance neededto regulate membrane potential, i.e., it plays an essentialrole in cell survival (Allen & Navran, 1984; Apell, 1989;Marín, 1993; Skou & Esmann, 1992). It is also thought to becritically involved in function such as cellular growth anddifferentiation and contraction of vascular smooth muscle,by virtue of its role in the maintenance of cellular ionic ho-meostasis (Allen & Navran, 1984; Apell, 1989; Marín,1993; Kaplan, 1985). The pump functions in a cyclic man-ner, transporting 2 mol of K

1

intracellularly for every 3 molof Na

1

moved extracellularly, and the energy for this trans-port is provided by the free energy of hydrolysis of 1 mol ofATP per cycle (Apell, 1989). The transport ratio 3Na

1

/2K

1

generates a potential across the membrane, being positiveon the outside (i.e., it is an electrogenic pump), which is es-sential for the cell (Skou & Esmann, 1992). Therefore, theinhibition of the Na

1

pump produces an ionic redistributionthat may underlie the increase in vascular tone (Blaustein,1977; Fleming, 1980; Hendrickx & Casteels, 1974; Marín etal., 1988; Van Breemen et al., 1979). This effect can be dueto an increase in intracellular Na

1

concentration, which atthe vascular smooth muscle level (myogenic component),causes an intracellular Ca

2

1

augmentation by decreasingCa

2

1

exit and/or by increasing Ca

2

1

entry through the Na

1

-Ca

2

1

exchange mechanism (Allen & Navran, 1984; Herms-meyer, 1983; Ozaki et al., 1978; Van Breemen et al., 1979)and membrane depolarization (Fleming, 1980; Hendrickx &Casteels, 1974), and at the level of adrenergic nerve end-ings, the release of noradrenaline (NA) (neurogenic compo-

nent) (Aarhus et al., 1983; Bonaccorsi et al., 1977; Marín etal., 1988; Palaty, 1980).

The Na

1

pump molecule consists of

a

- and

b

-subunits(Cantley, 1986; Schwartz & Adams, 1980); the transmem-brane

a

-subunit is responsible for most of the activities. Ithas an intracellular ATP hydrolytic site and an extracellulardigitalis glycoside-binding site (Cantley, 1986; Gick et al.,1993; Herrera et al., 1988; Horisberger et al., 1991; Jewellet al., 1992) (Fig. 1). At least 3 isoforms of the

a

-subunit(

a

1

,

a

2

, and

a

3

) and 2 of the

b

-subunit (

b

1

and

b

2

) in mam-mals have been described (Gick et al., 1993; Horisberger etal., 1991; Jewell et al., 1992); each isoform of the

a

-subunithas a different sensitivity to digitalis glycoside (Akera &Ng, 1991; Noël et al., 1990; Price & Lingrel, 1988). Hence,differences in the sensitivity of the vascular Na

1

pump tocardiac glycosides among species (Morita et al., 1988; Shi-bata et al., 1990) may be related to the respective isoformexpressed (Akera & Ng, 1991; Gick et al., 1993; Horis-berger et al., 1991; Noël et al., 1990; Price & Lingrel,1988). In addition, the isoform expressed in a tissue may bealtered in pathologic processes such as hypertension (Her-rera & Ruiz-Opazo, 1990) and probably also with age.

The endothelium seems to modulate the contractile re-sponse elicited by Na

1

pump inhibition by the release eitherof an unknown factor (Ponte et al., 1996a; Rodríguez-Mañas et al., 1994; Sánchez-Ferrer et al., 1993) or of endo-thelial nitric oxide (NO) (Arvola et al., 1992; Pörsti et al.,1992), which negatively modulates the response. The neuro-genic component is also modulated by an endothelial factorthat facilitates NA release (Rodríguez-Mañas et al., 1994).

The existence of endogenous ouabain-like factors that in-hibit Na

1

/K

1

-ATPase and their involvement in the develop-ment of high blood pressure has been controversial for

Fig. 1. Model of Na1/K1-ATPase structure. The a-subunit is proposed in this model to span the plasma membrane 8 times and to have both NH2- and COOH-termini in the cytoplasm. Phosphorylation of the enzyme by ATP or inorganic phosphate occurs on the amino acid Asp-369. The b-subunit spans the plasmamembrane a single time and has its NH2- and COOH-termini in the cytoplasm and the extracellular space, respectively. Three disulfide bridges within theb-subunit and three N-linked oligosaccharides of the b1-subunit isoform are shown.


251

many years (Goto et al., 1992; Goto & Yamada, 1998).However, some authors clearly support this hypothesis, pro-posing that these factors are indistinguishable from ouabain(Ferrandi et al., 1998; Hamlyn et al., 1991, 1996). In addi-tion, an elevated ouabain-like activity in response to Na

1

in-take has been reported in the spontaneous hypertensive rat(SHR) (Huang & Leenen, 1992).

The activity of the Na

1

pump may be altered in hyper-tension and diabetes. Thus, hypertension alters the re-sponses caused by pump inhibition (Moreland et al., 1986;Morita et al., 1988; Ponte et al., 1996a, 1996b; Shibata etal., 1990), as well as the endothelial modulation of such re-sponses (Ponte et al., 1996a, 1996b; Rodríguez-Mañas etal., 1994; Sánchez-Ferrer et al., 1993). Likewise, the activ-ity of this pump seems to be altered in diabetes (Bianchi etal., 1994; Garner, 1996; Scarpini et al., 1993).

The aim of the present study was to review the functionand properties of the vascular Na

1

pump, its regulation bythe endothelium, and its alterations in aging and pathologi-cal conditions, such as hypertension and diabetes.

2. Structure and isoforms of the Na

1

pump

The Na

1

/K

1

-ATPase is an integral membrane proteinpresent in most eukaryotic cells that directly or indirectlycontrols many essential cellular functions. This enzymecouples the energy released in the intracellular hydrolysis ofATP to the export of three intracellular Na

1

ions and theimport of two extracellular K

1

ions (Rose & Valdes, 1994).This transport produces both a chemical and an electricalgradient across the cell membrane that provides the energyfor the membrane transport of metabolites and nutrients,and it is essential also for regulation of cell volume and forthe action potential of smooth muscle and nerve (Lingrel &Kuntzweiler, 1994). This enzyme is a member of the P-typeclass of ATPases that shares a similar catalytic cycle in-volving a phosphorylated protein intermediate. In the caseof the Na

1

/K

1

-ATPase, the Asp-369 is the site that is phos-phorylated during the catalytic turnover of the enzyme (Fig.1) (Lingrel & Kuntzweiler, 1994). Limited site-directed mu-tagenesis studies have demonstrated that this residue is lo-cated within the large cytoplasmic globular domain of theenzyme (Ohtsubo et al., 1990). Phosphorylation is a keystep in the function of the Na

1

pump. The molecule under-goes an

a

-helix to

b

-sheet transition between two principalreactive states, E

1

and E

2

, in a multistep reaction by whichNa

1

ions and K

1

ions traverse the membrane (Jorgensen,1986; Rose & Valdes, 1994). The conformational transitionresults via a cyclic reaction in which the enzyme is phos-phorylated in the presence of Mg

2

1

ions and Na

1

ions andthen dephosphorylated in the presence of K

1

ions (Jor-gensen, 1986).

Recently, molecular techniques have revealed the entireprimary sequence of the Na

1

/K

1

-ATPase and have pro-vided new “working” membrane topology maps for the en-

zyme (Colonna et al., 1997; Pontiggia & Gloor, 1997; Rose& Valdes, 1994; Schneider & Scheiner-Bobis, 1997). Anti-body epitope localization studies indicate the position ofboth the NH

2

terminus (Felsengeld & Sweadner, 1988) andthe COOH terminus (Ovchinnikov et al., 1988) of the en-zyme on the cytoplasmic side of the plasma membrane (Fig.1). Four strongly hydrophobic amino acid sequences in theNH

2

-terminal region are predicted to cross the membrane as

a

-helices (H

1

–H

4

), while a middle third of the ATPase,which is not hydrophobic, is assumed to be folded as a glob-ular domain on the cytoplasmic surface of the membrane(Fig. 1) (Lingrel & Kuntzweiler, 1994). However, mem-brane topology models for the Na

1

/K

1

-ATPase often varyin the amino acid assignments with respect to the COOH-terminal region of the enzyme (H

5

–H

10

) (Canfield & Leven-son, 1993; Fisone et al., 1994). Therefore, discrepancies ex-ist among several current models of the Na

1

/K

1

-ATPase,and more data should be collected.

The Na

1

pump is composed of two subunits in anequimolar ratio. These are the

a

-subunit, with a molecularmass of

z

113 kDa, and the smaller glycosylated

b

-subunit,with a molecular mass of 55 kDa (Rose & Valdes, 1994),which have been cloned and sequenced (Kukreja et al.,1990; Vinnikova et al., 1992). The presence of a smaller

g

-subunit (10 kDa) has been suggested, but its role, if any,has not been well defined yet (Forbush et al., 1978; Rose &Valdes, 1994). The

a

-(catalytic) subunit contains all of thebinding sites both for ligands known to stimulate or inhibitthe enzyme and for ions (Daly et al., 1997; Farley et al.,1984; Karlish et al., 1990; Rogers & Lazdunski, 1979;Schneider & Scheiner-Bobis, 1997; Walderhaug et al.,1985). This subunit is proposed to have 7 (Ovchinnikov etal., 1987) or 8 (Fig. 1) (Shull et al., 1985) transmembranedomains; however, the number may vary from 6 to 10,since, as mentioned before, there are some discrepanciesbased on the interpretation of hydropathy profile data (Lin-grel et al., 1990) and identification of specific ligand-recep-tor interactions that predict

a

-chain topology (Karlish et al.,1991, 1993; Mohraz et al., 1994).

Three

a

isoforms of the Na

1

/K

1

-ATPase have been iden-tified, which are encoded by independent genes (Ovchinni-kov et al., 1987; Shull et al., 1986; Sweadner, 1991). The

a

1

isoform occurs in most tissues, while the

a

2

isoform is pre-dominant in skeletal muscle and is also detected in the brainand heart (Lingrel & Kuntzweiler, 1994; Rose & Valdes,1994). The

a

3

isoform is limited essentially to neural and car-diac tissue (Lingrel & Kuntzweiler, 1994; Rose & Valdes,1994). The three isoforms have the same molecular weight,but there are differences in their amino acid compositionand sequence (Lingrel & Kuntzweiler, 1994). The sequenceidentity is about 82%, with the major differences in theN-terminal region (Skou & Esmann, 1992). They differ intheir sensitivity towards cardiac glycosides, and their ex-pression depends on the developmental stage and/or tissuestudied (Rose & Valdes, 1994). Moreover, isoform specific-ity even extends to cell type within a particular tissue (Mar-

252


tin-Vasallo et al., 1989; Sweadner, 1992; Zahler et al.,1992), supporting the hypothesis that the isoforms have dif-ferent physiological functions (Jewell & Lingrel, 1992; Mc-Donough et al., 1992).

Structure studies on the

a

-subunit indicate that the bind-ing of ouabain takes place from the extracellular side of thatsubunit, although labeling and site-directed mutagenesisstudies have demonstrated that the binding site is composedof multiple functional groups, and the loss of any one doesnot completely prevent binding (Burns & Price, 1993; Can-essa et al., 1992; Feng & Lingrel, 1994; Price & Lingrel,1988; Price et al., 1989, 1990; Schultheis & Lingrel, 1993).Nevertheless, the majority of the substitutions that affectouabain binding and/or sensitivity are found between thefirst and second transmembrane segments of the

a

-subunit(Lingrel & Kuntzweiler 1994). Thus, it has been suggestedthat there exists a crucial correlation between ouabain affin-ity differences and the isoform type expressed based on the

a-subunit structure (Sweadner, 1989). Hence, differences inthe sensitivity of the vascular Na1 pump to cardiac glyco-sides among species, for example, the low sensitivity of ratblood vessels to the Na1 pump inhibitor ouabain (Morita etal., 1988; Shibata et al., 1990), may be related to the respec-tive isoform expressed (Akera & Ng, 1991; Gick et al.,1993; Horisberger et al., 1991; Noël et al., 1990; Price &Lingrel, 1988). The Na1 pump isoform expression differ-ences could also have relevant therapeutic implications insome pathological situations, such as hypertension and heartfailure, in which there is altered isoform expression (Her-rera et al., 1988; Herrera & Ruiz-Opazo, 1990; McDonoughet al., 1995). However, the physiological importance andsignificance of this hypothesis are still scarcely explored.

Three Na1 pump b isoforms and one related H1/K1-ATPase b (b4) isoform have been identified. The predominantform in mammalian kidney is b1, which has been isolatedfrom several vertebrate species in a wide range of tissues(Rose & Valdes, 1994; Skou & Esmann, 1992). The b2-sub-unit is expressed largely in brain (Martin-Vasallo et al.,1989; Shyjan et al., 1990) and seems to play a dual role,both as a subunit of the Na1 pump and as a mediator of cell-cell interaction (Gloor et al., 1990). The third isoform, b3,has been isolated from Xenopus (Good et al., 1990), and fi-nally, the H1/K1-ATPase b4-subunit has been isolated andcloned from rabbit and rat (Sweadner, 1991). The sequencesof b-subunits reveal a low sequence similarity of about 40%between these isoforms. All of them have a single hydro-phobic transmembrane domain and are heavily glycosylatedon the extracellular side, with a mass of sugar of about 10kDa and some disulfide bridges (Fig. 1) (Esmann et al.,1980). The role of the b-subunit is not very clear, but it ap-pears to be involved in maturation of the enzyme, its attach-ment to the plasma membrane, and stabilization of a K1-bound intermediate form of the protein (Eakle et al., 1994;Geering, 1990, 1991; Lutsenko & Kaplan, 1993; McDon-ough et al., 1990). It is known that cellular expression of theb-subunits and assembly with the a-subunits are necessary

for correct conformation and activity of the Na1/K1-ATPase holoenzyme (McDonough et al., 1990). Moreover, itseems that the extracellular domain of the Na1 pump b iso-forms mediates the interactions with the a1-subunit and in-fluences the stability of this complex (Pontiggia & Gloor,1997). Its participation in ion translocation is still uncertain,but the presence of the b-subunit appears essential for thefunction of the Na1 pump (McDonough et al., 1990).

In vascular tissues, the function of the Na1 pump is totransport Na1 out of the cell and K1 into the cell (Fleming,1980; Hermsmeyer, 1982; O’Donnell & Owen, 1994). Thevascular Na1 pump structure is similar to that found in othertissues, but with some particularities. The enzyme of vascu-lar tissue has been difficult to isolate and purify using con-ventional protein purification techniques, due to a low num-ber of copies of the pump in these cells (Allen et al., 1986).Nevertheless, the importance of the Na1 gradient in bloodpressure regulation has long been recognized (Friedman etal., 1958), and many studies have been carried out to char-acterize the enzyme and its role in modulating vascular con-traction (Allen et al., 1986; Hendrickx & Casteels, 1974;Lang & Blaustein, 1980). Since the discovery that both a-and b-subunits of the Na1 pump exist in different isoformsin other tissues, as mentioned before, the existence of thesemultiple isoforms for the vascular enzyme has also beensuspected and is supported by experimental evidence(Weiss et al., 1993). Sahin-Erdemli et al. (1994) have de-scribed the existence of a1-, a2-, and a3-subunit proteins (allz97.5 kDa) in cultured rat aortic smooth muscle cells andrat tail arteries by Western-blot techniques. In addition tothese a forms, they detected a minor band at about the 68-kDa position in aortic smooth muscle cells, which may cor-respond with the truncated a1 protein reported earlier byMedford et al. (1991). It has been postulated that the pres-ence of these small structural differences in the ATPasemay permit variations in the responsiveness and regulationof Na1 pump function between vascular smooth muscle andother tissues (Allen et al., 1989). Moreover, it recently hasbeen described that endothelial cells from human umbilicalvein express b1 and b2 isoforms, a large amount of a1 iso-form with an apparently low affinity for ouabain, and alesser amount of high-affinity sites, which may correspondto the a3 protein (Mayol et al., 1998). In addition, since thevascular Na1 pump plays a key role in modulating bloodpressure, the regulation of its activity has been extensivelystudied. Experimental evidence has shown that the vascularenzyme can be stimulated by treatments that increase intra-cellular Na1 concentration and/or decrease intracellular K1

concentration (O’Donnell & Owen, 1994). Brock and Smith(1982a) demonstrated that the ionophore monensin in-creased total cell Na1 and subsequently, ouabain-sensitive86Rb1 uptake (a measure of Na1 pump activity) in culturedsmooth muscle cells; an effect that was not observed in theabsence of external Na1. Additional evidence demonstratedthat the activation of the vascular Na1 pump can occur viahormone-induced elevation of intracellular Na1 (Fig. 2)

J. Marín, J. Redondo / Pharmacology & Therapeutics 84 (1999) 249–271 253

(Brock et al., 1982; Navran et al., 1988, 1991; Smith &Brock, 1983). More recent studies have also shown that theincrease in intracellular Na1 by mechanical strain (Liu etal., 1998; Songu-Mize et al., 1996) or chronic treatmentwith ouabain (Liu & Songu-Mize, 1998) up-regulates thea1- and a2-isoform expression of the Na1 pump in aorticsmooth muscle cells. In other tissues, phosphorylation ofthe a-subunit of the Na1 pump by protein kinases C (PKC)and A (Fig. 2) (Bertorello et al., 1991; Chibalin et al., 1992)produces a decrease in its activity; however, whether thevascular Na1 pump is regulated via a mechanism involvingphosphorylation of the a-subunit in a similar way remainsto be clarified.

3. Na1 pump and vascular tone

The sarcolemmal Na1/K1-ATPase plays an essentialrole in the maintenance of vascular smooth muscle tone(Haddy, 1983; Marín et al., 1988; Van Breemen et al., 1979;Vasalle, 1987; Webb & Bohr, 1978). Thus, the inhibition of

this enzyme by cardiac glycosides or K1-free solutions pro-duces an increase in vascular tension in several vessels(Maigaard et al., 1985; Marín et al., 1988; Ozaki &Urakawa, 1979; Sato & Aoki, 1988; Toda, 1980; Wallick etal., 1982). The reported cellular mechanisms involved in thecontraction after the Na1 pump blockade are different de-pending on the vascular bed and the animal species. Thiscontraction can be produced by (1) a direct effect on vascu-lar smooth muscle (myogenic component) mediated by de-polarization and opening of the voltage-operated Ca21 chan-nels (VOCCs) in the vascular smooth muscle (Fig. 2)(Fleming, 1980; Hendrickx & Casteels, 1974; Vasalle,1987) or Ca21 entry into these cells through the inversion ofthe Na1-Ca21 exchange system (Fig. 2) (Blaustein, 1988;Casteels et al., 1985; Fernández-Alfonso et al., 1992b;Marín et al., 1991; Ozaki et al., 1978; Ozaki & Urakawa,1979) and (2) NA release (neurogenic component) after in-hibition of the Na1/K1-ATPase in the perivascular nerveendings (Fig. 3) (Broekaert & Godfraind, 1973; Karaki &Urakawa, 1977; Rodríguez-Mañas et al., 1994). The pre-dominant component depends on the animal used, the kind

Fig. 2. Scheme of signal transduction pathways in vascular smooth muscle cells for short-term regulation of Na1 pump activity and the mechanisms involvedin the Na1 pump inhibition-evoked vasoconstriction by ouabain or endogenous digitalis-like factors. Activation of membrane receptors linked to adenylylcyclase (AC) by hormones, such as a2 and b agonists, alters cyclic AMP (cAMP) levels, which activates protein kinase A (PKA) and subsequently cPLA2,resulting in the hydrolysis of membrane phospholipids (P-lipids) and the production of arachidonic acid (AA) and PGE2, potent inhibitors of the Na1 pump.Other hormones, such as angiotensin II, endothelin-1, and 5-HT, may activate the phospholipase C (PLC), generating inositol trisphosphate (IP3) and DAG, apotent activator of the PKC. PKC activation may inhibit or stimulate the Na1 pump activity. Atrial natriuretic peptide (ANP) and NO increase cyclic GMP(cGMP) levels through the bound- (GC) or soluble-guanylyl cyclase (SCG), which in turn activate the cGMP-kinase, whose role in Na1 pump activity regula-tion is not very clear. Finally, an increase in intracellular Na1 levels by hormones or mechanical strain produces a direct stimulation of the vascular Na1

pump. Na1 pump inhibition by ouabain or endogenous digitalis-like factors increases intracellular Na1 concentration, which causes Ca21 entry by the inver-sion of the Na1-Ca21 exchange mechanism and/or by depolarization and opening the VOCCs. Gi, Gs, and Gp are G proteins.

254 J. Marín, J. Redondo / Pharmacology & Therapeutics 84 (1999) 249–271

of vessel studied, and the density of adrenergic innervation(Bonaccorsi et al., 1977; Hayashi & Park, 1984; Marín etal., 1988; Ponte et al., 1996a; Toda, 1980; Wallick et al.,1982). It is interesting to comment that the rat vascular Na1

pump is very resistant to inhibition by cardiac glycosides, ashigh concentrations of these compounds (0.1-1 mM oua-bain) are needed to observe a clear vascular response (Mo-reland et al., 1986; Morita et al., 1988; Ponte et al., 1996a;Shibata et al., 1990).

In human umbilical arteries, ouabain causes a rapid tran-sient (early) contraction and a sustained slow (late) contrac-tion (Sato & Aoki, 1988). However, in human placental ar-teries and veins, digoxin (Maigaard et al., 1985) or ouabain(Fernández-Alfonso et al., 1992b; Marín et al., 1991) onlyproduce slowly developed contractions, which are not bi-phasic. These responses are totally due to the myogenic ac-tion of glycosides because umbilicoplacental vasculaturelacks adrenergic innervation (Reilly & Russell, 1977; Walker& McLean, 1971). In the rat aorta, the neurogenic partici-pation in the contraction after the Na1 pump blockade is

also very weak (Ponte et al., 1996a). In cat cerebral arteries,ouabain induces transient contractions, whereas in cat femo-ral arteries, contractions are sustained (Marín et al., 1988).

The extracellular Ca21 dependence of the responses elic-ited by Na1 pump inhibition with cardiac glycosides cur-rently has been reported, as the responses are abolished inCa21-free medium (Ashida & Blaustein, 1987; Fernández-Alfonso et al., 1992b; Marín et al., 1988; Ozaki & Urakawa,1981; Toda, 1980). However, some authors reported con-tractions to glycosides in a medium without Ca21 (Maigaardet al., 1985; Sato & Aoki, 1988). This disagreement can beexplained by the concentration of EGTA (to complex extra-cellular Ca21) in the Ca21-free solution, which in the lattercase, contains a lower EGTA concentration (0.1 mM or 10mM) than in the former (1 mM), and probably the extracel-lular Ca21 was not completely removed.

The contribution of VOCCs to the ouabain effect hasbeen suggested by the electrogenic action of the Na1/K1-ATPase, blockade of which can originate a small cell depolar-ization (Fleming, 1980; Hendrickx & Casteels, 1974; Va-salle, 1987). This quick depolarization precedes any signifi-cant change in the intracellular Na1 concentrations, but canbe sufficient for opening VOCCs, originating the contractileresponse (Fig. 2) (Fleming, 1980; Hendrickx & Casteels,1974; Vasalle, 1987). However, the capacity of the cardiacglycosides to depolarize different vessels is variable, de-pending on the contribution of the Na1/K1-ATPase to themembrane potential of the respective smooth muscle cells(Harder et al., 1983; Marín et al., 1988). The participation ofthese Ca21 channels in the contractile responses to cardiacglycosides has been examined by using the Ca21 agonistBay K 8644 and Ca21 antagonists. The results obtained byvarious authors suggest that this mechanism scarcely partic-ipates in these responses, but other mechanisms differentfrom depolarization, which were observed in several vascu-lar preparations, including placental and umbilical vesselsdo participate in these responses (Bova et al., 1988; Fernán-dez-Alfonso et al., 1992b; Maigaard et al., 1985; Marín etal., 1991; Ozaki & Urakawa, 1979; Sato & Aoki, 1988; VanBreemen et al., 1979). However, the early transient contractileresponse induced by ouabain in umbilical arteries is sensitiveto the Ca21-channel blockade (Sato & Aoki, 1988) and alsothe contraction induced by this agent in cat cerebral arteries(Marín et al., 1988).

The most frequent mechanism involved in the contrac-tion caused by Na1 pump inhibition is Ca21 entry into thesmooth muscle cells by the inversion of the Na1-Ca21 ex-change system described in different vascular beds (Fig. 2)(Ashida & Blaustein, 1987; Bova et al., 1988; Fernández-Alfonso et al., 1992b; Johansson & Hellstrand, 1987; Marínet al., 1991; Ozaki & Urakawa, 1979, 1981).

4. Methods to characterize the vascular Na1 pump

Different methods have been used to characterize thevascular Na1 pump: K1-induced relaxations, measurement

Fig. 3. Mechanisms involved in the contractions elicited by ouabain orendogenous digitalis-like factors acting on an endothelial cell (EC). Neuro-genic (NA release) and myogenic (direct effect on the Na1 pump ofsmooth muscle cell, SMC) components of the contractions are shown. Bothcomponents are mediated by the release of two probably different factors:F1, which stimulates or protects the SMC Na1 pump, and F2, which inhibitsthe Na1 pump of the noradrenergic nerve endings. The inhibition of theNa1 pump in sympathetic nerve endings and the SMC produces Na1 accu-mulation and Ca21 entry (ion involved in vascular contraction and NArelease) through Na1-Ca21 exchange. This Na1 pump blockade producesNA release, which activates postsynaptic a-adrenoceptors. Furthermore,ouabain inhibits NA reuptake.


of 32P release, ouabain-sensitive 86Rb1 uptake, and [3H]oua-bain binding to arterial membrane fractions or vascularsmooth muscle cells.

4.1. K1-induced relaxation

An indirect method classically used for measuring theactivity of the vascular Na1 pump is to incubate vascularsegments in a K1-free medium from a period of time andthen contract the segments with an agonist, such as NA or5-hydroxytryptamine (5-HT). The subsequent addition ofK1 to the bath produces a relaxation by activation of theNa1 pump that induces hyperpolarization and a Ca21 influxblockade (Bonaccorsi et al., 1977; Fernández-Alfonso et al.,1992a, 1992c; Hermsmeyer, 1983; Marín et al., 1988). Themagnitude of this relaxation is an index of Na1 pump activ-ity (Marín et al., 1988; Webb & Bohr, 1978), as it is inhib-ited by ouabain. Thus, the fact that this relaxation washigher in cat cerebral arteries than in femoral arteries sug-gests a higher activity of this pump in the former arteries(Marín et al., 1988). This medium produces a reversible in-hibition of the Na1 pump (Haddy, 1983; Hermsmeyer,1983; Van Breemen et al., 1979) and usually a contractileresponse (Bonaccorsi et al., 1977; Hayashi & Park, 1984;Marín et al., 1988), as occurs with cardiac glycosides. Nor-mally, this response is lower than that caused by ouabain forunknown reasons (Fernández-Alfonso et al., 1992a, 1992c;Hayashi & Park, 1984; Marín et al., 1988).

4.2. Measurement of 32P release

Na1/K1-ATPase activity may be measured by the re-lease of 32P in an enzymatic reaction with [g-32P]ATP(Kelly et al., 1985; Tao et al., 1994). Since the activity ofthe enzyme and the number of copies of the pump in vesselsare very low in comparison with other tissues such as heart(Marín et al., 1988; O’Donnell & Owen, 1994), it is amethod that is not very useful in vessels. In addition, differ-ent conventional protein extraction and purification tech-niques have been used to isolate the vascular enzyme, whichmight explain the contradictory results obtained with thismethod (Manjeet & Sim, 1987).

4.3. Ouabain-sensitive 86Rb1 uptake

86Rb1 is an analogue of K1 and is used by the cells simi-larly to K1. It has the advantage of a considerably longerhalf-life (18 days vs. 12 hr for 42K1), which permits bettermanagement in the laboratory (Bukoski et al., 1983;Fernández-Alfonso et al., 1992a, 1992c; Marín et al., 1988),for studies of arterial segments (Marín et al., 1988) and ofsmooth muscle cells (Redondo et al., 1995, 1996). There-fore, transport activity mediated via the Na1 pump has beenfrequently assayed as ouabain-sensitive 86Rb1 influx or up-take. However, although this technique is very useful to de-termine the activity of the enzyme, the comparison between

results from different studies is quite difficult, since 86Rb1

uptake is dependent on the time of incubation with the ra-dioisotope. In the case of cat cerebral and femoral arteries,ouabain-sensitive uptake was higher in cat cerebral arteries,which agrees with the results obtained by K1-induced relax-ation, as mentioned in Section 4.1 (Marín et al., 1988).

4.4. [3H]Ouabain-binding experiments

A number of investigators have assessed the number ofNa1 pump sites by evaluating specific binding of [3H]oua-bain to smooth muscle preparations (Deth & Lynch, 1980;Fernández-Alfonso et al., 1992a, 1992c; Gerthoffer & Allen,1981; Marín et al., 1988) and vascular smooth muscle cells(Khalil et al., 1984; Redondo et al., 1995). Analyses of[3H]ouabain binding to a given vessel preparation can becomplicated by (1) a low number of receptor sites for oua-bain, (2) poor binding of the glycoside, and (3) a high per-centage of nonspecific binding sites. However, it is stillconsidered a very useful method and has been used exten-sively, as much in vascular tissue as in cultured cells. UsingScatchard analysis of the results, it is possible to determinethe Kd, the affinity (1/Kd) of the Na1 pump for ouabain, andBmax. Thus, Kd values were similar in cat cerebral (5 nM)and femoral (8.4 nM) arteries (Marín et al., 1988). Thesevalues resemble those obtained in microsomes of dog me-senteric arteries (ranging from 2 to 9 nM) (Adams et al.,1983; Wallick et al., 1982) and bovine aorta (4 nM) (Fox etal., 1983), but lower than those reported in human placentalarteries (88.3 nM; Fernández-Alfonso et al., 1992c) andveins (196.7 nM; Fernández-Alfonso et al., 1992a), suggest-ing a different affinity in these vessels. Bmax values are303.5 and 74 fmol/mg of protein in cat cerebral and femoralarteries (Marín et al., 1988), respectively, and 345 and 1606fmol/mg in human placental arteries (Fernández-Alfonso etal., 1992c) and veins (Fernández-Alfonso et al., 1992a), re-spectively, whereas in the dog mesenteric artery (Adams etal., 1983; Wallick et al., 1982) and bovine aorta microsomes(Fox et al., 1983), the Bmax values range from 2 to 6.24pmol/mg. In cat heart membranes, the Kd and Bmax valueswere 14.2 nM and 6560 fmol/mg (Marín et al., 1988). Theseresults indicate that the affinity for ouabain is similar in catcerebral and femoral arteries and heart, but the number ofNa1 pump sites in vascular smooth muscle is less than incardiac muscle. This fact agrees with the elevated density ofNa1 pump units widely described for heart in comparisonwith vessels (Adams et al., 1982; Finet et al., 1982). In cul-tured vascular smooth muscle cells from normotensive andhypertensive rats, the Kd values are quite similar and in amicromolar range, which is indicative of the poor bindingof ouabain to its receptors in these preparations (Khalil etal., 1984; Redondo et al., 1995). However, such a high Kd

value is not unique to these cells; for instance, Kd valuesranging between 1027 and 1025 M have also been docu-mented for the rat myocardium (Adams et al., 1982; Finet etal., 1982).


5. Na1 pump and endothelial modulation on noradrenaline release

Previously, it has been indicated that cardiac glycosidesmay produce contraction by NA release from the perivascu-lar sympathetic nerve endings (neurogenic component)(Fig. 3). In addition, the increase in NA concentration in theneuromuscular cleft by these drugs may also be due to theinhibition of the Na1 pump, which produces a reduction inthe NA uptake into the sympathetic nerve endings (Haddy,1990). The NA release by these glycosides has been sug-gested by different authors to be caused by membrane depo-larization and Ca21 influx through VOCCs (Wallick et al.,1982). However, others suggest it to be due to an increase inCa21 concentration in adrenergic nerve endings by the Na1-Ca21 exchange as a consequence of the Na1 pump inhibi-tion in these terminals (Adams et al., 1983; Magyar et al.,1987; Marín et al., 1986). Our group demonstrated that suchNA release from densely sympathetically innervated ves-sels, such as guinea-pig carotid artery (Rodríguez-Mañas etal., 1994), is facilitated by endothelial release of a diffusiblefactor(s), the nature of which is unknown (Fig. 3). The ex-istence of this diffusible factor was demonstrated by bioas-say (Rodríguez-Mañas et al., 1994). This factor is neitherNO nor a prostaglandin (PG)-related compound, as con-firmed by the lack of effect of the NO blocker oxyhemoglo-bin and the cyclooxygenase blocker indomethacin (Ro-dríguez-Mañas et al., 1994).

The influence of the endothelium on ouabain-evoked NAliberation has also been directly observed by the ability ofthe glycoside to release tritium from guinea-pig carotid ar-teries preincubated with [3H]NA. Ouabain produced time-dependent tritium secretion in a way similar to that reportedin other vessels after Na1 pump blockade (Bonaccorsi et al.,1977; Marín et al., 1986; Palaty, 1980). This ouabain-induced radioactivity release was significantly reduced inde-endothelialized arteries, in agreement with the resultsobtained in vascular reactivity. This was the first report toshow that neurogenic contraction elicited by ouabain can bemediated by the endothelium, although inhibitory effects ofendothelial cells on adrenergic neurotransmission have alsobeen described (Cohen & Weisbrod, 1988; Greenberg et al.,1989; Tesfamariam et al., 1987; Tesfamariam & Cohen,1988). These latter effects have been attributed to NO pro-duction by the endothelium, a mechanism not involved inouabain-induced NA release.

6. Na1 pump and vasomotor responses

6.1. Na1 pump and endothelium-dependent relaxation

Na1 pump inhibition with cardiac glycosides or K1-freesolution not only produces contractile responses with myo-genic and neurogenic components (see Section 3), but caninhibit endothelium-dependent relaxation (De Mey & Van-houtte, 1980; Rubanyi & Vanhoutte, 1985, 1988). It is known

that acetylcholine and other vasodilator agents can releaseNO and endothelium-derived hyperpolarizing factor (EDHF).The latter causes smooth muscle hyperpolarization and sub-sequently closes Ca21 entry through Ca21 channels (Boltonet al., 1984; Chen et al., 1988; Feletou & Vanhoutte, 1988;Huang et al., 1988; Keef & Bowen, 1989; Komori & Su-zuki, 1987; Komori et al., 1988; Marín & Sánchez-Ferrer,1990). In low-resistance arteries, the EDHF appears to be amajor determinant of vascular calibre under normal condi-tions and, therefore, may be of primary importance in theregulation of vascular resistance (Garland et al., 1995; Quil-ley et al., 1997). It has been proposed that the mechanism ofaction of EDHF could be the stimulation of the Na1/K1-ATPase activity in vascular smooth muscle cells, based onthe inhibition of the hyperpolarizing responses by Na1

pump blockade (Cohen & Vanhoutte, 1995; Feletou & Van-houtte, 1988). Indeed, ouabain reduces acetylcholine-evoked vasodilation in the fetal rat aorta due to EDHF re-lease (Martínez-Orgado et al., 1999). Moreover, it has beensuggested that Na1 pump blockade might cause a depolar-ization that would inhibit the hyperpolarizing effect on K1

channels modifying endothelium-dependent relaxation (Taylor& Weston, 1988). Despite these findings, some authors havereported that EDHF actions are not related to Na1 pump ac-tivity (Chen et al., 1989; Suzuki, 1988).

The fact that endothelium-dependent relaxation was me-diated by Na1 pump activation, at least in part, can be ofphysiological relevance by its possible implication in patho-logical events such as hypertensive disease, in which a cir-culating factor that blocks Na1/K1-ATPase activity hasbeen detected (De Wardener et al., 1987; Hamlyn et al.,1982; Overbeck, 1987). This effect is associated with theexistence of alterations of the endothelium and the abnor-malities of endothelium-dependent responses observed inhypertension (Marín & Sánchez-Ferrer, 1990; Marín & Ro-dríguez-Martínez, 1997; Panza et al., 1990).

The inhibition of endothelium-dependent relaxation bycardiac glycosides can also be mediated by the blockade ofthe endothelial NO effect (Boulanger et al., 1989; Hoeffneret al., 1989) or by interference with the endothelial synthe-sis or release of NO (Woolfson & Poston, 1991). Recently,the ability of ouabain to reduce the hyperpolarizationcaused by adenosine receptor-mediated hyperpolarization inporcine coronary artery smooth muscle, which is partiallyendothelium-dependent and possibly involved in the Na1

pump activation and NO release, has been demonstrated(Olanrewaju et al., 1997).

6.2. Na1 pump and endothelium-independent relaxation

The vasorelaxation caused by b-adrenergic agonists maybe mediated, at least partially, by Na1 pump activation(Somlyo et al., 1970; Webb et al., 1981). This mechanismhas been observed in rat and pig tail (Webb & Bohr, 1981)and canine coronary arteries (Cooke et al., 1988). It seemsthat the enhancement of intracellular cyclic AMP by b-adren-


ergic stimulation, or by agents that increase this cyclic com-pound, hyperpolarizes the smooth muscle cells by activatingthe Na1 pump, as reported in different arteries (Somlyo etal., 1970, 1972), and can stimulate Na1/K1-ATPase of mi-crosomal fractions from dog mesenteric arteries (Limas &Cohn, 1974). Likewise, intracellular cyclic GMP may alsohave the ability to activate vascular Na1/K1-ATPase (Alonsoet al., 1993; Rapoport & Murad, 1983; Rapoport et al.,1985b), which can be associated with the vasodilation in-duced by sodium nitroprusside, which releases NO in the rataorta (Rapoport & Murad, 1983; Rapoport et al., 1985a), catfemoral arteries (Alonso et al., 1993), and rabbit coronaryand femoral arteries (Foley, 1984b). Furthermore, other va-sodilators also have the capacity to activate the Na1 pumpthat mediates, at least in part, their response. This is the caseof PGs in pig coronary (Fukuda et al., 1992), rat tail (Lock-ette et al., 1980; Webb et al., 1981), and dog mesenteric ar-teries (Limas & Cohn, 1974); the diffusible cyclic analogue8-bromo-cyclic GMP (Rapoport et al., 1985a); adenosine inhuman coronary (Sabouni & Mustafa, 1989) and rabbit cor-onary and femoral arteries (Foley, 1984a, 1984b); atrialnatriuretic factor in the rabbit aorta (Sybertz & Desiderio,1985); and dihydropyridine calcium antagonists in tail arter-ies (Hermsmeyer & Rusch, 1988) and the rat aorta (Pan &Janis, 1984). However, sodium nitroprusside, 8-bromo-cy-clic GMP, and PGs do not activate the Na1 pump in cul-tured smooth muscle cells of Wistar-Kyoto (WKY) rats andSHRs (Redondo et al., 1995).

6.3. Influence of the Na1 pump on agonist-induced contractions

The participation of Na1/K1-ATPase in the contractileresponses elicited by different agonists has been reported(Bell et al., 1989; Fernández-Alfonso et al., 1992a, 1992c;Moreland et al., 1985; Navran et al., 1991). It is interestingto comment on its influence on the potent vasoconstrictionelicited by 5-HT. Thus, in dog and rabbit mesenteric arteries(Bell et al., 1989; Moreland et al., 1985) and human placen-tal arteries and veins (Fernández-Alfonso et al., 1992a,1992c), the increase in the Na1 pump activity in response to5-HT has been proposed to be due to the increase in mem-brane Na1 permeability induced by the amine. Some inves-tigators have also demonstrated that the vascular Na1 pumpactivation by 5-HT is produced through a PKC-dependentmechanism (Fig. 2) (Navran et al., 1991). Other vasocon-strictor substances, such as angiotensin II (Brock & Smith,1982b; Smith & Brock, 1983), a-adrenergic agonists (Nav-ran et al., 1988), and endothelin (Gupta et al., 1991), havebeen shown to activate the vascular Na1 pump by elevationof intracellular Na1. In cultured rat aortic smooth musclecells from WKY rats and SHRs, angiotensin II and endothe-lin-1 increased ouabain-sensitive 86Rb1 uptake more in hy-pertensive- than in normotensive-derived cells (Redondo etal., 1995), suggesting a possible alteration in the regulationof the enzyme by these vasoconstrictors in cells from the

hypertensive strain. Furthermore, depolarization producedby the increase in intracellular Na1 concentration may alsosensitize the vascular smooth muscle to vasoconstrictoragents, such as NA (Haddy, 1990; Hinke, 1965). In thissense, the oral administration of digoxin appears also to in-crease vascular reactivity to NA and angiotensin II (Guthrie,1984; Mikkelsen et al., 1979a, 1979b; Zemel & Sowers,1990).

Human placental arteries possess a significant populationof Na1/K1-ATPase units (Marín et al., 1991), and the inhi-bition or stimulation of this enzyme markedly affects thevascular tone of these vessels (Fernández-Alfonso et al.,1992c). The importance of 5-HT in the umbilicoplacentalcirculation is supported by its elevated concentrations (z0.1mM) in umbilical blood (Jones & Rowsell, 1973; O’Reilly& Loncin, 1967). It seems that Na1/K1-ATPase can exertan important role in the regulation of umbilicoplacental re-activity to this amine, especially in pregnancy-induced hy-pertension, a condition in which researchers (Delva et al.,1989; Graves, 1987; Kelly et al., 1985; Odendaal et al.,1986) have detected in circulating blood the presence of in-hibitors of this pump that might increase vascular resistanceto placental blood flow.

7. Endothelial modulation on the vascular Na1 pump

7.1. Arterial segments

It is known that the endothelium plays an important rolein the regulation of vasoconstrictor and vasodilator re-sponses elicited by different agonists (Angus & Cocks,1989). Therefore, it is possible that the contractile responseselicited by Na1 pump inhibition with cardiac glycosideswere modulated by factor(s) released from the endothelium.Our group has demonstrated that the contractile effects ofouabain (which seems to be an endogenous digitalis-likesubstance; see Section 9) in the guinea-pig carotid artery(Rodríguez-Mañas et al., 1992; Sánchez-Ferrer et al., 1993),placental vessels (Sánchez-Ferrer et al., 1992, 1993), andaorta from SHRs and WKY rats (Ponte et al., 1996a, 1996b;Sánchez-Ferrer et al., 1993) seem to be modulated by the re-lease of an endothelial unknown and bioassayable factor(s)(Fig. 3); this factor is neither a PG nor an NO-related com-pound, as it was unaltered by inhibitors of cyclooxygenaseand NO synthase, respectively. Moreover, in pressurizedmiddle cerebral arteries from SHRs and WKY rats, ouabainreduces relaxations that were positively modulated by en-dothelium, which releases a factor distinct from NO(González et al., 1999). However, some authors have ob-served that the endothelial factor involved in these effectsappears to be NO in rat mesenteric arteries (Arvola et al.,1992). Also, the NA release induced by ouabain and in-volved in its contractile responses is positively modulatedby the release of an unknown endothelial factor(s), as previ-ously noted (Rodríguez-Mañas et al., 1994). This meansthat in hypertension, in which endothelium is altered (Marín,


1993; Marín & Rodríguez-Martínez, 1997; Panza et al.,1990), the responses induced by ouabain may be modified,as reported (Ponte et al., 1996a, 1996b). Therefore, it is pos-sible that the loss of a protective effect of endothelium onNa1 pump activity could facilitate the vascular Na1/K1-ATPase inhibition by endogenous circulating digitalis. It isworth noting that the contractile effects of cardiac glyco-sides in human vessels are due mainly to direct actions ofthese agents on smooth muscle cells (Mason et al., 1986;Mikkelsen et al., 1979a, 1979b; Woolfson et al., 1990), bywhich the possible antagonistic effects of the endotheliummay have a higher physiological role. Recently, it has beenreported that NO released from NO donors (as sodium ni-troprusside) may inhibit the Na1 pump purified from por-cine cerebral cortex by interacting with an SH group at theactive site of the enzyme (Sato et al., 1995). This mecha-nism is not involved in the effects of ouabain described byour group in guinea-pig carotid arteries, placental arteries,and rat aorta, previously noted, since endothelial NO is notreleased by Na1 pump inhibition. In the guinea-pig isolatedtrachea, contractions induced by ouabain are also potenti-ated by epithelium removal (Raeburn & Fedan, 1989). Twopossibilities have been proposed to explain this effect: (1)the blockade by the glycoside of a relaxant factor(s) re-leased from epithelial cells and (2) the secretion by the epi-thelium of a factor(s) able to stimulate the activity of the un-derlying smooth muscle Na1 pump (Raeburn & Fedan, 1989).

7.2. Cultured vascular smooth muscle cells

As noted in Section 7.1, the endothelium of vascular seg-ments releases an endothelial factor that negatively modu-lates the response elicited by Na1 pump inhibition. The ex-istence of this factor is more directly analyzed by using co-cultures of aortic vascular smooth muscle cells and endothe-lial cells from bovine aorta (Redondo et al., 1995, 1996).Basal activity of this pump in vascular smooth cells fromWKY rats was similar to SHRs measured by ouabain-sensi-tive 86Rb1 uptake (Redondo et al., 1995). In co-cultures for24 hr of endothelial cells and vascular smooth muscle cells,or using conditioned medium of endothelial cells, Na1

pump activity was increased in smooth muscle cells, this ef-fect being higher in hypertensive-derived cells (Redondo etal., 1995). Analogous enhancement of Na1 pump activity invascular smooth muscle cells by co-cultured endotheliumpreviously has been reported by Berk (1989). The condi-tioned medium obtained in the presence of inhibitors of an-giotensin-converting enzyme, endothelin-1 converting en-zyme, cyclooxygenase, lipoxygenase, and NO synthase didnot modify the Na1 pump activation. Therefore, angiotensinII, endothelin-1, eicosanoids (Orlov et al., 1992; Paquet etal., 1990), or NO (Arvola et al., 1992), which have been re-ported to activate the Na1 pump, are not involved in thisstimulatory effect (Redondo et al., 1995).

The nature of the possible diffusible endothelial factorinvolved is still unknown, but it possesses a molecular mass

between 25 and 50 kDa, is heat stable at 1008C for 3 min,but not 10 min, and is sensitive to trypsin treatment. It wasproposed to be a growth factor (Redondo et al., 1995), asendothelial mitogens can stimulate the Na1 pump in cul-tured vascular smooth muscle cells (Allen et al., 1989; Berk,1989) and may have contractile or relaxant properties (Berket al., 1985; Cunningham et al., 1992). The mechanism in-volved in the stimulation of the Na1 pump by conditionedmedium of endothelial cells has been analyzed by ourgroup. This mechanism is mediated by stimulation of PKCin vascular smooth muscle cells from either WKY rats orSHRs, although the SHR cells show some alterations intheir intracellular signaling pathways (Redondo et al.,1996). Recently, it has been reported that this protein is anendogenous regulator of pulmonary artery endothelial cellNa1 pump activity (Charles et al., 1997).

8. Cardiac glycosides and nitric oxide production

As noted in Section 6.1, the inhibition of the Na1 pumpby ouabain causes an impairment of endothelium-dependentrelaxation in different vascular preparations by decreasedrelease of NO (Woolfson & Poston, 1991) or by the block-ade of its effect (Boulanger et al., 1989; Hoeffner et al.,1989). However, it has been described that ouabain is ableto selectively enhance basal release of NO from the vascularendothelium of the porcine carotid artery (Xie et al., 1993).Moreover, some authors have shown that ouabain providesa complementary signal for either tumor necrosis factor orinterferon-g to increase the expression of the NO synthaseinducible form in murine endothelial cells (Beretta et al.,1995). In vascular smooth muscle cells, the inducible NOsynthase activity is also induced by cytokines and endotox-ins (Koide et al., 1993) and plays an important role in vas-cular contractility, as well as vascular inflammation (Diner-man et al., 1993; Ikeda et al., 1995). An increased NOproduction by an inhibition of the Na1 pump with ouabainhas also been shown in vascular smooth muscle cells stimu-lated with interleukin-1-b (Ikeda et al., 1995). In a similarway, ouabain enhanced the lipopolysaccharide-induced NOproduction by rat peritoneal macrophages (Sowa & Przew-locki, 1997) and mouse motor nerve terminals (Liu et al.,1995). In all cases, ouabain caused no effect on basal re-lease of NO, but significantly stimulated NO synthesis un-der cytokine- or endotoxin-stimulated conditions.

Although the mechanism of ouabain-induced enhance-ment of cytokine- or endotoxin-induced NO production isunknown, increased expression of “de novo” inducible NOsynthase seems to be involved in this effect (Beretta et al.,1995; Ikeda et al., 1995). In addition, since Ca21 iono-phores, such as ionomycin or A23187, mimicked the effectof ouabain on NO production, some authors have speculatedthat ouabain might be acting via increases of intracellularCa21 ion concentrations (Ikeda et al., 1995; Sowa & Przew-locki, 1997). In cultured vascular smooth muscle cells, a


ouabain-induced depolarization seems to be more likely in-volved in this effect on [Ca21]i; this effect may produceCa21 influx that may be abolished by VOCC blockers (Mul-vany & Aalkjaer, 1985; Woolfson et al., 1990; Zhu et al.,1994).

NO production by the inducible NO synthase activity inthe blood vessel walls and vascular smooth muscle cells hasbeen implicated in different pathophysiological conditions,such as sepsis-related systemic hypotension, atherosclero-sis, and hypertension (Garg & Hassid, 1989; Joly et al.,1992; McNamara et al., 1993; Wennmalm, 1994). There-fore, the effect of cardiac glycosides on NO synthesis mightbe of importance in the above conditions in vivo, and fur-ther studies are needed to clarify these points.

9. Physiological role of endogenous digitalis-like factors

Since cardiac glycosides were suggested as endogenousphysiological regulators of heart muscle contraction (Buck-alew & Haddy, 1989), there has been a long search for natu-ral endogenous ligands of the Na1 pump and many studiesabout its involvement in the maintenance of high bloodpressure in hypertension. It has been hypothesized that theobserved increase of peripheral resistance in hypertensioncould be partially due to increased plasma levels of a hu-moral circulating factor that inhibits the Na1 pump(Blaustein, 1996; Goto et al., 1992; Haddy & Pamnani,1998; Schoner, 1991). The humoral factor theory is espe-cially applicable to low-renin, salt-sensitive hypertensivepatients who have a defect in Na1 excretion (Buckalew &Haddy, 1989). The endogenous digitalis is thought to be in-volved in Na1 homeostasis and blood pressure regulation asa vasoactive and natriuretic compound (Goto et al., 1991;Haddy & Pamnani, 1998). When Na1 ingestion exceeds thecapacity of kidney excretion, an expansion in the plasmavolume occurs, which can be sensed by the central circula-tion, leading to the release of atrial natriuretic peptide andmainly of digitalis-like substance(s), both inhibiting theNa1 pump (Abbott, 1988; Haddy, 1990) and sensitizingblood vessels to vasoconstrictor agents (Haddy, 1990; Plun-kett et al., 1982). Thus, the inhibition of the Na1 pump inthe proximal tubules produces natriuresis, whereas the inhi-bition of that present in sympathetic nerves and vascularsmooth muscle cells causes an elevation in blood pressure;this elevation is caused by an increase in both catechola-mine secretion and vascular tone (Abbott, 1988; Haddy,1990), and subsequently an increased contractility (Buck-alew & Haddy, 1989). Therefore, the Na1 pump inhibitionwould induce an increased natriuresis and would protectblood volume at the expense of an elevated blood pressure(Blaustein, 1977; Goto et al., 1992; Haddy & Overbeck,1976; Haddy, 1987; Poston, 1987).

Na1 pump inhibition has been described in cells (Ed-mondson et al., 1975), vessels (Huot et al., 1983; Overbecket al., 1976; Pamnani et al., 1978, 1981a, 1981b; Songu-

Mize et al., 1982), and myocardium (Clough et al., 1983,1984; Haddy, 1990) of animals and patients with low-reninhypertension. Moreover, decreased ouabain-sensitive Na1/K1-ATPase activity with increased intracellular Na1 con-centration has been described in erythrocytes, leukocytes,and lymphocytes from hypertensive and normotensive pa-tients with a family history of hypertension (Ambresioni etal., 1981; Cooper et al., 1987; De Wardener & Macgregor,1983; Edmondson et al., 1975; Heagerty et al., 1982).

Some authors have suggested that the digitalis-like sub-stance could also participate in the maintenance of hyper-tension. Indeed, in some patients with heritable essential hy-pertension and in hypertensive animal models such as one-kidney, deoxycorticosterone acetate (DOCA)-saline ratsand SHRs, the basic alteration in blood vessels could be anaugmented membrane permeability to Na1 (Buckalew &Haddy, 1989). The addition to this defect of an endogenousfactor, after Na1 ingestion, would potentiate the intracellu-lar Na1 concentration and would reduce Na1 excretion, pro-ducing and maintaining hypertension (Haddy, 1990). Insome cases, chronic administration of digoxin or ouabain toanimals was accompanied by increases in blood pressure(Spence et al., 1989; Yuan et al., 1993) and mild hyperten-sion (Overbeck, 1985). Yuan et al. (1993) showed thatchronic administration of low doses of ouabain induced hy-pertension in normotensive rats, as well as rats with variousdegrees of reduced renal mass. Probably the association ofouabain with excessive DOCA-saline treatment might beincreasing the hypertensive effect of ouabain. This assump-tion has been demonstrated in mononephrectomized ratstreated with ouabain in combination with DOCA-salinetreatment (Sekihara et al., 1992).

Numerous studies have been reported showing elevatedcirculating digitalis-like activity in hypertensive subjects(Aalkjaer & Mulvany, 1983; Abel & Hermsmeyer, 1981;Alaghband-Zadeh et al., 1983; Ayachi & Brown, 1980;Buckalew et al., 1987; Buckalew & Haddy, 1989; Goto &Yamada, 1998). Goto et al. (1990) showed that the presenceof plasma from hypertensive subjects reduces the [3H]oua-bain binding to tubular renal cells to a greater extent thanwith plasma from normotensive subjects; this suggests theexistence of a digitalis-displacing compound in plasma,whose levels were enhanced in patients with essential hy-pertension. In addition, incubation of platelets from normalsubjects with plasma from hypertensive patients produced amarked (80%) enhancement in platelet Ca21 levels. How-ever, when platelets from hypertensive patients were incu-bated with plasma from normotensive subjects, this resultedin a 33% reduction in platelet Ca21, suggesting the exist-ence of a circulating factor (likely the endogenous digitalis-like substance) partially responsible for augmentation in in-tracellular Ca21 (Lindner et al., 1987; Zemel & Sowers, 1990).

Although so far no substance has convincingly beenidentified as the endogenous digitalis-like factor, strong evi-dence suggests that the major biological source for thatcompound is the human circulation and the bovine hypo-


thalamus (Goto & Yamada, 1998). The factor seems to be asteroidal isomer of ouabain (ouabain-like compound)(Blaustein, 1996; Ferrandi et al., 1998; Hamlyn et al., 1996)in which the location and orientation of two or more steroi-dal hydroxyl groups differ (Hamlyn et al., 1998). This oua-bain-like compound has been found in human urine andneonatal cord blood (Balzan et al., 1997; Kramer et al.,1998; Worgall et al., 1996) and it is present in elevated cir-culating levels in humans with essential hypertension(Hamlyn et al., 1998). An endogenous digoxin-like com-pound has also been identified with structural characteris-tics similar to those of digoxin and has been isolated fromthe adrenal cortex of humans and cows (Qazzaz et al., 1996)and from human urine (Goto & Yamada, 1998). Other po-tential digitalis-like substances are still being identified. In-deed, it appears that mammals might also have bufodieno-lides, which have been detected in human lenses (Samuelov& Lichtstein, 1997), bovine and rat adrenals (Lichtstein etal., 1998), and plasma from volume-expanded subjects (Fe-dorova et al., 1998; Wright et al., 1997). Another digitalis-like substance distinct from ouabain has also been isolatedfrom volume-expanded renal failure patients, whose levelscorrelate closely with the volume status and blood pressureof the patients (Tao et al., 1996). Therefore, several inde-pendent lines of evidence show the existence of differentdigitalis-like substances, although their nature, structure,and physiological implications in hypertension remain to beclarified.

10. Na1 pump and hypertension

Some aspects concerning the relationship between theNa1 pump and hypertension have been discussed in Section9. Hypertension is a disease that provokes important alter-ations in the cardiovascular system and supposes a more el-evated risk of cardiovascular events in the elderly than inyounger people (Marín, 1995; Rose & Valdes, 1994). Alter-ations of the endothelium (Marín, 1993; Marín & Ro-dríguez-Martínez, 1997), Ca21 movements, and Na1 trans-port (Marín, 1993) have been described in this disease. TheNa1 pump is a major cellular transport system that controlsNa1 homeostasis (O’Donnell & Owen, 1994) and mem-brane potential (Hermsmeyer & Erne, 1990), both key fac-tors in the regulation of vascular tone and blood pressure(Marín et al., 1988; Marín, 1995), which suggests that an al-tered Na1 pump could be an underlying factor in essentialhypertension (Blaustein et al., 1986; Brock et al., 1982;Hermsmeyer, 1982, 1983; Magargal & Overbeck, 1986;Magliola et al., 1986; Wong et al., 1984). Indeed, alterationsof electrogenic ion transport for membrane potential at restin SHRs have been shown by Hermsmeyer (1976a, 1976b,1982). An increased Na1 pump activity has been describedin mineralocorticoid-salt hypertension (Magliola et al.,1986), chronic DOCA salt, or one-kidney Grollman renalhypertension (Brock et al., 1982). Such augmented enzy-

matic activity in hypertension could be explained by an in-crease in passive permeability to Na1 that is not fully com-pensated by the pump and thus, vascular tone is elevated(Friedman et al., 1975; Jones & Warden, 1980). However,other authors have shown a decreased myocardial and vas-cular Na1 pump activity in different models of hypertension(Clough et al., 1983; Pamnani et al., 1981b), suggesting thata reduced pump activity might be common in low-renin hy-pertension, either by an inherent defect or by a circulatinginhibitor of the pump (a digitalis-like compound), whichleads to augmented intracellular Na1 levels and thus, an in-creased vascular tone (Blaustein et al., 1986). In addition,changes in Na1 pump activity during hypertension may bedue to alterations in Na1 pump unit and/or isoform expres-sion (O’Donnell & Owen, 1994). Indeed, a specific increasein Na1 pump a1-subunit mRNA and increased expression ofthe pump unit number by elevation of intracellular Na1 lev-els produce a sustained elevation of the pump activity (Krug& Berk, 1992). Alterations in the content of a1, a2, and a3

isoform mRNA expression in the aorta, heart, and skeletalmuscle of rats made hypertensive by DOCA and salt treat-ment (Herrera et al., 1988) have also been demonstrated. At theprotein level, an increased a1 isoform and a decreased a2-subunit expression have been reported in heart membranes(Sahin-Erdemli et al., 1995) with no alteration in skeletalmuscle or the whole brain (Sahin-Erdemli et al., 1995) dur-ing DOCA-salt hypertension. In this latter case, the modi-fied Na1 pump expression did not alter the activity of theenzyme (Sahin-Erdemli et al., 1995). Further studies aboutisoform-specific differential regulation of expression duringhypertension are needed to clarify the roles of the isoformsand the molecular mechanisms involved in hypertension.

On the other hand, altered endothelial modulation of theNa1 pump during hypertension has been shown by ourgroup in aortic segments (Ponte et al., 1996a) and pressur-ized middle cerebral arteries (González et al., 1999) ofWKY rats and SHRs. In intact aortic segments, the contrac-tile response to ouabain was higher in WKY rats than inSHRs (Ponte et al., 1996a). However, an increase in theseresponses in SHR arteries compared with WKY rat arterieshas been reported (Moreland et al., 1986; Morita et al.,1988; Sakai & Inazu, 1991; Shibata et al., 1990). These au-thors suggest that such higher responses can be due to en-hanced Na1 pump activity, a higher Na1-Ca21 exchange, orother alterations in the membrane permeability to Na1 andCa21 (Marín, 1993; Moreland et al., 1986; Morita et al.,1988; Sakai & Inazu, 1991; Shibata et al., 1990), or even tothe existence of vascular hypertrophy associated with hy-pertensive disease (Schwartz et al., 1990). In stroke-proneSHR tail arteries, the relaxation caused by K1 (7 mM) addi-tion was greater in hypertensive than WKY rats (Rinaldi &Bohr, 1989). These researchers reported that this increase inNa1 pump activity by hypertension may be due to enhancedNa1 leakiness of hypertensive smooth muscle (Rinaldi &Bohr, 1989). In renal hypertensive rats, but not in normoten-sive rats, ouabain increases the contractions elicited by phe-


nylephrine and KCl in the rat aorta, an effect blocked bythapsigargin (Ceron & Bendhack, 1997), an inhibitor of sar-coplasmic reticulum Ca21-ATPase. Thapsigargin did not al-ter basal tone, but increased it in the presence of ouabain.These authors suggest that enhanced cytoplasmic Na1 con-centration by ouabain produces Ca21 accumulation in hy-pertensive rats, a mechanism to modulate cytosolic Ca21 inrenal hypertension (Ceron & Bendhack, 1997).

The endothelium seems also to modulate the response toouabain in a different way in WKY rat and SHR segments,probably influenced by endothelial dysfunction associatedwith hypertensive disease, as reported (Marín, 1993; Marín& Rodríguez-Martínez, 1997). Endothelium removal increasesand reduces ouabain-induced contractions in WKY rat andSHR aortic segments, respectively, suggesting a negative inWKY rat and positive in SHR endothelial modulation ofouabain contractions (Ponte et al., 1996a). The negative en-dothelial modulation of ouabain response obtained in nor-motensive WKY rat aorta has also been described in humanchorionic vessels (Sánchez-Ferrer et al., 1992), guinea-pigcarotid arteries (Rodríguez-Mañas et al., 1992), and in WKYrat mesenteric arteries with a K1-free solution (Arvola et al.,1992; Pörsti et al., 1992), as discussed in Section 7.1. Thesecontradictory results between WKY rats and SHRs suggestrelease of two different factors from the endothelium ofboth strains: an endothelial contractile factor that potenti-ates ouabain contractions and another that stimulates Na1

pump activity (Ponte et al., 1996a). In the case of WKY rataortic segments, the endothelial substance that activates thepump would predominate over the contractile factor (Ponteet al., 1996a). In the contractility experiments in the SHRaorta, however, the endothelial facilitation of ouabain-induced contractions appears to predominate (Ponte et al.,1996a). This assumption agrees with data reported by otherauthors, who suggest that the loss of endothelium-mediatedrelaxations in the SHR vasculature is due to increased en-dothelium-dependent contractions that mask the effects ofvasodilator factors (Iwama et al., 1992; Lüscher & Van-houtte, 1986; Lüscher et al., 1988).

Na1 pump activity, measured as ouabain-sensitive 86Rb1

uptake, was reduced in SHR vessels in comparison withWKY rat vessels (Ponte et al., 1996a), whereas other au-thors describe an increased activity of the vascular Na1/K1-ATPase in SHRs (Cox, 1986; Pamnani et al., 1981a; Webb& Bohr, 1979). Probably these authors did not take into ac-count the modulator role of the endothelium on the vascularNa1 pump activity, since a reduction of this activity hasbeen shown when the endothelium is considered (Arvola etal., 1992; Pörsti et al., 1992). The activity of the Na1 pump,measured by K1-induced relaxation and ouabain-sensitive86Rb1 uptake, seems to be decreased in small mesenteric ar-teries and splanchnic veins of renal hypertensive dogs(Overbeck et al., 1976). These authors suggest that this ab-normality may partially depolarize the vascular smoothmuscle and may explain the elevated vascular resistance ob-served in these dogs. However, impaired K1-induced relax-

ation has been described in pressurized rat cerebral arteriesfrom SHRs, which is not a consequence of Na1 pump alter-ation, but of Na1 pump-independent mechanisms (McCar-ron & Halpern, 1990). By enzymatic measurement of Na1

pump activity in the aorta smooth muscle and endotheliumfrom SHRs, it was observed that this activity is reduced inhypertension compared with WKY rats (Manjeet & Sim,1987). These authors comment that blood pressure increasemay be related to an alteration in cation transport across theplasma membrane. In addition, these authors review the in-fluence of hypertension on Na1 pump activity, and an in-crease, decrease, and no alteration has been reported. Thisvariability may be dependent on the models of hyperten-sion, the type of vessel, and the method used for Na1 pumpactivity determination (Manjeet & Sim, 1987).

The existence of an endothelial factor that facilitates oua-bain-induced contraction in SHRs was demonstrated by ourgroup in bioassay experiments (Ponte et al., 1996a, 1996b).Thus, when the bioassay ring was from SHR aorta and thedonor segment from WKY rat aorta, a negative endothelialmodulation of ouabain-evoked contractions was observed,similar to that observed when both bioassay ring and donorsegment were from WKY rats. In contrast, when the bioas-say ring was from WKY rats and the donor segment fromSHRs, facilitation of ouabain-induced vasoconstrictionswas observed, i.e., a phenomenon similar to that obtainedwhen both segments came from SHRs. A more detailedstudy of this phenomenon showed that the endotheliummight activate and/or partially protect the vascular smoothmuscle Na1/K1-ATPase from ouabain blockade in bothstrains (Ponte et al., 1996a), although in SHRs, an endothe-lial contractile factor seemed also to be involved.

In aortic cultures of smooth muscle cells from SHRs orWKY rats, basal Na1 pump activity was similar in bothstrains (Redondo et al., 1995). Endothelial cells co-culturedwith vascular smooth muscle cells or conditioned mediumfrom the endothelium produced an increase in smooth mus-cle Na1 pump activity from WKY rats and SHRs, this acti-vation being higher in SHRs (Redondo et al., 1995, 1996).This finding agrees with previous results reported in cellcultures by Orlov et al. (1992), but disagrees with the in-creased Na1 pump activity reported in SHR-derived vascu-lar smooth muscle cells by other authors (Kuriyama et al.,1992; Tamura et al., 1986). The reason for such discor-dances is not entirely clear, but this fact could be reflectingdifferent growing states of vascular smooth muscle cell cul-tures. Some authors use subconfluent proliferating cultures(Kuriyama et al., 1992), while confluent nonproliferatingvascular smooth muscle cell cultures were employed by ourgroup (Redondo et al., 1995, 1996) and other researchers(Orlov et al., 1992). However, the number of Na1/K1-ATPase units per milligram of cellular protein (Bmax), as wellas its affinity for ouabain, was similar in vascular smoothmuscle cell cultures from both WKY rats and SHRs (Redondoet al., 1995). Therefore, there was no difference in either thenumber of Na1 pump sites and/or activity in confluent non-


proliferating cultures from normotensive- and hypertensive-derived cells (Redondo et al., 1995), although the regulationof its activity seems to be significantly different (Redondoet al., 1995).

11. Diabetes and Na1 pump activity

Diabetes has a marked effect on the metabolism of a va-riety of tissues, including vascular smooth muscle and en-dothelium (Arnqvist, 1977; Cohen, 1993, 1995). The patho-genesis of vascular disease in diabetes may involveaccelerated atherosclerosis, thrombosis, hypertension, andhyperlipidemia (Cohen, 1993). Because the Na1 pump iscritical for the membrane potential, growth, differentiation,and contraction of vascular smooth muscle (Kaplan, 1985),a change in its activity in diabetes would have profoundconsequences in vascular tissues. A decrease in Na1 pumpactivity has been reported in aorta (Ohara et al., 1991; Smithet al., 1997), carotid (Tesfamariam et al., 1993), and mesen-teric arteries (Smith et al., 1997) of diabetic animals. Thisdecrease in Na1 pump activity has been attributed to a de-pletion of the intracellular pool of myo-inositol, increasedflux through the aldose reductase pathway, and an alterationin PKC activity (Greene et al., 1987).

The biochemical mechanism by which elevated glucoselevels reduce Na1 pump activity has been the topic of multi-ple studies, but the results have been confusing concerningthe role of PKC (Vasilets & Schwarz, 1993). Recently, ithas been shown that the inhibition of the Na1 pump is dueto an activation of PKC induced by hyperglycemia in vascu-lar cells (Xia et al., 1995; King et al., 1996). Since 1990, ithas been known that elevated levels of glucose increase thelevel of diacylglycerol (DAG), which in turn activates PKC,specifically the PKC bII isoform, in cultured vascular cellsand many vascular tissues (Inoguchi et al., 1992, 1994; Kai-ser et al., 1992; Xia et al., 1994). Hyperglycemia- or diabe-tes-induced increases in DAG and PKC levels have beenfound in a variety of vascular cells in culture, as well as invascular tissues, from diabetic animals and patients (King etal., 1996). When the PKC b isoforms are activated by hy-perglycemia, the cytosolic phospholipase A2 (cPLA2) activ-ity becomes activated, as demonstrated by both increasedphosphorylation of cPLA2, as well as its activity and theproduction of arachidonate (Nishio et al., 1995). Activationof cPLA2 is also associated with increased PGE2 production(King et al., 1996). Since both PGE2 and arachidonate arepotent inhibitors of the Na1 pump (Fig. 2) (Schwartzman etal., 1985), it has been proposed that this is the mechanismby which hyperglycemia inhibits the Na1 pump (King et al.,1996). This hypothesis is further supported by the fact thatPKC and cPLA2 inhibitors are able to normalize the inhibi-tory effect of hyperglycemia on Na1 pump activity (King etal., 1996; Kowluru et al., 1998; Koya et al., 1997).

As discussed in Section 7, it has been demonstrated thatthe endothelium normally has a stimulatory action on theNa1 pump in the blood vessels (Berk, 1989; Ponte et al.,

1996a, 1996b; Redondo et al., 1995; Rodríguez-Mañas etal., 1992; Sánchez-Ferrer et al., 1992, 1993). An impairedendothelial relaxation of the rabbit aorta subjected to highglucose concentrations (Tesfamariam et al., 1991) has beenshown. Similar results were obtained with aortic rings fromdiabetic rabbits (Tesfamariam et al., 1989). Furthermore,the release of endothelium-derived factors is altered by hy-perglycemia (Jeremy et al., 1983; Tesfamariam et al., 1989,1991; Yamaguchi et al., 1990). Since the endotheliumseems to be impaired in diabetes (Cohen & Tesfamariam,1992; Cohen, 1993), endothelial modulation of the vascularNa1 pump could also be modified. Indeed, inhibition ofNa1 pump activity has been seen in endothelium-intact aor-tas from severely diabetic rabbits (Gupta et al., 1992; Sim-mons & Winegrad, 1991) or normal aortas incubated inhigh glucose solutions (Gupta et al., 1992; Simmons &Winegrad, 1989). Diabetic arteries, or those incubated in el-evated glucose, have decreased Na1 pump activities similarto normal arteries from which the endothelium has been re-moved or that were treated with an inhibitor of NO synthase(Cohen, 1993; Gupta et al., 1992). The Na1 pump activityin arteries exposed to elevated glucose is restored to normalby incubating the artery with L-arginine, a precursor of NOsynthesis, or sodium nitroprusside (Cohen, 1993; Gupta etal., 1992). These findings suggest that a defect in NO syn-thesis occurs in diabetic blood vessels, which in turn im-pairs the Na1 pump in the vascular smooth muscle (Cohen,1993). Since the Na1 pump regulates the membrane poten-tial and the contractile tone, as well as the endothelium-dependent relaxation, a reduced pump activity may help toexplain impaired vascular responses in diabetes (Cohen, 1993).

On the other hand, hypertension is frequently associatedwith insulin-dependent diabetes mellitus (Arnqvist, 1977;Wertheimer & Ben-Tor, 1962). It has been reported that di-abetes caused by streptozotocin may produce hypertensionin the presence of impaired renal function (Chen et al.,1993). Plasma levels of a digoxin-like immunoreactive fac-tor are increased in association with a decrease in the myo-cardial Na1 pump (Chen et al., 1993). These authors sug-gest that an endogenous digitalis-like substance could play arole in the pathogenesis of insulin-dependent diabetes melli-tus hypertension by inhibiting cardiovascular muscle cellNa1 pump activity (Chen et al., 1993).

12. Changes in the Na1 pump elicited by aging

There are few studies in the literature that analyze the in-fluence of age on Na1 pump activity. Our group has studiedthis influence in normotensive and hypertensive rats. Thus,experiments achieved in aortic segments from WKY ratsand SHRs of different ages (5 weeks and 3, 6, 12, and 18months) show that aging markedly enhances contractile re-sponses to ouabain in both strains, although their evolutionis quite different between WKY rats and SHRs (Ponte et al.,1996b). Some hypotheses have emerged to explain the in-crease in the response to ouabain with age, such as an in-


crease in the expression of Na1 pump high-affinity isoforms(a2 and a3) for cardiac glycosides (Jewell et al., 1992; Ng &Akera, 1987, 1991; Ng & Book, 1992) or an alteration ofmembrane permeability to Na1 and Ca21 (Kennedy & Sei-fen, 1988).

Changes in endothelium functionality caused by aginghave been described in different vascular beds, character-ized by a reduction in endothelium-dependent relaxationsand an increase in endothelium-dependent contractions(Koga et al., 1989; Hongo et al., 1988; Küng & Lüscher,1995). In accordance with this, our results suggest that thedifferences in contractile responses to ouabain with agecould be caused, at least partially, by changes in the proper-ties of the vascular endothelium. Thus, the endotheliumnegatively modulated ouabain contractions in young (3–6months) WKY rats, whereas it facilitated ouabain responsesin segments from old (18 months) normotensive rats andfrom 3- to 12-month-old SHRs (Ponte et al., 1996b). Thesedata might be consistent with the assumption that hyperten-sion produces a premature endothelial dysfunction, andmight suggest that the aortic endothelium of young normo-tensive rats might release a factor that negatively modulatesouabain contractions, activating the Na1 pump (Ponte et al.,1996b). The existence of such a factor has been discussed inSection 7. On the other hand, the facilitation of ouabain re-sponses by endothelium in old (18 months) normotensiverats and 3- to 12-month-old hypertensive rats may be due tothe release of an endothelium-dependent contracting factorin a way similar to that reported for acetylcholine in aorticsegments (Iwama et al., 1992; Kato et al., 1990; Lüscher &Vanhoutte, 1986). Therefore, the loss of the negative endo-thelial modulation of ouabain-evoked contractions observedin hypertension and aging could be due to the existence orprevalence of an endothelium-contracting factor that potenti-ates ouabain contractions in these states (Ponte et al., 1996b).

Alterations or even no modification of Na1 pump activ-ity in aging have been reported. Indeed, an aging-associatedreduction in Na1 pump activity has been shown in bothcrude ventricular homogenates and partially purified sar-colemmal preparations (Katano et al., 1984; Kendrick et al.,1980). In aortic segments from 6- and 8-month-old WKYrats and SHRs, aging reduced Na1 pump activity, measuredby K1-induced relaxations, in normotensive rats, but had noeffect in hypertensive rats (Ponte et al., 1996b). Results ob-tained in mesenteric and cerebral arteries from beagles indi-cated that Na1 pump activity is unaltered by aging (Toda etal., 1986). However, an age-related increase in Na1 pumpactivity has been observed in rabbit basilar arteries (Toda &Hayashi, 1979). These results suggest that aging may altervascular Na1 pump activity, likely depending on the vesselsand/or animal species studied.

13. Concluding remarks

The importance of the vascular Na1 pump in the regula-tion of vascular tone and its alteration in some diseases and

aging have been described. Although excellent progress hasbeen made in the understanding of the structure-function ofthe Na1 pump and its regulation, many questions still re-main open. The knowledge of the regulation of the pumpactivity in molecular terms still represents a formidable sci-entific challenge. The description of the vascular Na1 pumpprimary structure and the identification of tissue-specificisoforms open a new field for different approaches to designand development of a new generation of cardiotonic and an-tihypertensive drugs.

How the contraction evoked by vascular Na1 pump inhi-bition with cardiotonic steroids is related to the existence ofmultiple Na1 pump a-subunit isoforms has been investi-gated recently (Blaustein et al., 1998). It has been proposedthat the Na1 pump high-affinity (a3) isoform and the Na1-Ca21 exchanger are confined to membrane domains thatoverlie junctional sarcoplasmic reticulum in vascularsmooth muscle, while low ouabain affinity a1 is uniformlydistributed in the membrane (Blaustein et al., 1998). Thus,low doses of cardiotonic steroids, including endogenousdigitalis-like factors, may influence cytosolic Na1 and (in-directly) Ca21 concentrations only in the cytoplasmic cleftsbetween the membrane and junctional sarcoplasmic reticu-lum (Blaustein et al., 1998). This new hypothesis could ex-plain the cardiotonic and vasoactive effects of low doses ofcardiotonic steroids that do not elevate cytosolic Na1 orCa21. Moreover, this novel idea may provide new light onthe mechanism of action of cardiotonic steroids and mayhelp to explain how endogenous digitalis-like factors canwork effectively as hormones.

In the future, a continued investigation into the vascularNa1 pump and its alteration in pathological processes even-tually might improve the efficacy of hypertension and dia-betes treatments and reduce morbidity and mortality fromthese prevalent diseases.

Acknowledgments

This work was supported by grants from D.G.I.C.Y.T.(PM97-0008), Comunidad de Madrid (08.3/0002/1997),FIS (98/0074-02), and Bayer España. We thank Ms. Ana B.Baena for her kind collaboration in the preparation of themanuscript.

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