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Environmental Health Perspectives 

Vol. 78, pp. 113-115,1988 

Effects of Lead and Natriuretic Hormone 

on Kinetics of Sodium-PotassiumActivated Adenosine Triphosphatase: 

Possible Relevance to Hypertension 

by Elmar Weiler,* Farhad Khalil-Manesh,* and 

Harvey Gonick^ 

Inhibition of vascular smooth muscle sodium-potassium-activiated adenosine triphosphatase (NaK-ATPase) has been postulated as a central mechanism in enhancing vascular contractility. In the 

present study, kinetics of inhibition of Na-K-ATPase by lead, ouabain, and natriuretic hormone (NH) 

was studied in a purified hog cerebral cortex enzyme preparation. Determination of I50 values for 

lead, ouabain, and NH revealed that NH is the most potent inhibitor of the enzyme system (0.8 x 

IO"6 M ouabain equivalents). Kinetic analyses indicated that lead and NH exhibited different 

inhibitory mechanisms. The inhibition by lead was noncompetitive with respect to potassium and 

competitive with respect to sodium and MgATP. Natriuretic hormone was noncompetitive with 

respect to potassium, uncompetitive with respect to MgATP, and exhibited no inhibitory effect with 

respect to sodium. Synergism between lead and NH in the inhibition of Na-K-ATPase raises the 

possibility that lead may be a contributory factor in hypertension via this mechanism. 

Introduction 

Epidemiological studies performed both in the 

United States (1) and in Great Britain (2) have suggested a correlation between blood pressure and blood 

lead within the range of blood lead levels seen in the 

normal population. The prevalence of hypertension in 

industrially exposed workers has been a matter of 

controversy. Studies performed by Cramer and 

Dahlberg in 1966 (3) showed no difference in the 

incidence of hypertension in workers directly exposed 

to lead as compared to age-matched workers in the same 

plant who had minimal lead exposure. More recent 

studies by de Kort et al. (4) and Kirby et al. (5) 

demonstrated a positive relationship between blood 

lead and blood pressure in lead-exposed populations. 

Thus, the issue has been raised that lead may be a possible causative or contributory factor in hypertension. 

Lead, like many other heavy metals (vanadium, 

•Trace Element Laboratory, Division of Nephrology/Department of 

Medicine, Cedars-Sinai Medical Center and School of Medicine, 

University of California, Los Angeles, CA 90048. 

tAuthor to whom reprint requests should be addressed. 

mercury, cadmium, uranium) (6-9) is a potent inhibitor of the membrane transport enzyme sodiumpotassium-activated adenosine triphosphatase (Na-KATPase, E. C. 3.6.1.3.). This effect of lead, if exerted on 

vascular smooth muscle, might produce enhanced 

vascular contractility. Within the past few years, there 

has been considerable interest in the possibility that a 

circulating transport enzyme inhibitor [the putative 

hypothalamic-renal natriuretic hormone (NH)] 

might be a major cause of essential hypertension via 

the same mechanism (10-12). A hypothesis advanced 

by Blaustein et al. (11) is that inhibition of the vascular smooth muscle sodium pump by. NH leads to 

increased intracellular calcium concentrations, thus 

enhancing vascular responsiveness to the vasoconstrictive hormones norepinephrine and angiotensin 

II. 

If lead and NH could be shown to inhibit Na-KATPase by different mechanisms, then the possibility 

exists that lead might act to increase vascular contractility by synergizing with NH in inhibiting the 

vascular smooth muscle sodium pump. The goals of 

the present study were first to contrast the kinetics of 

inhibition of highly purified Na-K-ATPase by lead 

J 
P.3 

114 WEILER, KHAULMANESH, AND GONICK 

and a semi-purified plasma Na-K-ATPase inhibitor, 

(NH), with the known specific inhibitor of Na-KATPase, ouabain, and second to explore the possibility 

of synergistic inhibition of the enzyme by lead and 

NH. 

Methods 

Purification of Natriuretic Hormone 

from Human Plasma 

Separation of low molecular weight NH from high 

molecular weight material was achieved by passing 

the plasma sample from a hypertensive patient 

through an Amicon YM 2 (1 kilodalton cutoff) membrane at 20 psi. For further purification of the low 

molecular weight material, the filtrate was passed 

through a SEP-PAK cartridge and eluted with 

acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) 

as described in an earlier publication (13). The fraction 

eluting between 0% ACN and 20% ACN was lyophilized 

and stored for kinetic studies. Concentration of 

inhibitor was expressed as ouabain-equivalents (13). 

Lead acetate used for the kinetic studies was purchased 

from Fisher Scientific (Fair Lawn, NJ). 

Na-K-ATPase Inhibition Assay 

In these experiments a purified Na-K-ATPase from 

hog cerebral cortex was employed, purchased from 

Sigma Chemical (St. Louis, MO). The incubation 

tubes contained a substrate solution providing final 

concentrations of 1 mM ATP; 1 mM Mg; 10 mM 

imidazole-HCl buffer, pH 7.2; 100 mM Na; 20 mM K; 

and 1 mM ethylene glycol-bis-((3-aminoethyl ether) 

NyN' tetraacetic acid (EGTA). The tubes were incubated 

at 37°C for 5 min. The reaction was started by adding 

0.025 mL of enzyme preparation (25 mg/mL). The 

incubation was stopped after 10 min by adding ice-cold 

trichloracetic acid (TCA). After centriiugation (HOOgx 

5 min), 0.5 mL of supernatant was assayed for 

inorganic phosphate by the method of Fiske and 

Subbarow (14). In all instances, enzyme inhibition 

was expressed as jimole Pi/mg protein/hr. 

For determination of I50, i.e., the amount of 

inhibitor necessary for 50% enzyme inhibition, serial 

dilutions of the purified low molecular weight plasma 

NH were prepared. For comparison, ouabain and lead 

were dissolved in water to result in final concentrations ranging from 4 x IO"8

 M to 4 x 10"3 M ouabain 

and 5 x IO-5 M to 5 x 10^ M Pb. 

To further characterize the inhibitory activities of 

lead, ouabain, and NH, kinetic studies were performed 

with concentrations of potassium ranging from 0.125 

mM to 5.0 mM, sodium ranging from 5 mM to 120 

mM, and MgATP ranging from 0.25 mM to 3.0 mM, 

in the presence of constant amounts of lead, ouabain, 

and NH. For the MgATP studies, the ratio between Mg 

and ATP molar concentrations was maintained at 1:1. 

Michaelis-Menten parameters were calculated from the 

conventional Lineweaver-Burk plot. 

Synergy 

In order to determine whether lead and NH were 

synergistic inhibitors of Na-K-ATPase, a lead doseresponse curve was first established. The combined 

effects of lead and NH were assayed in the presence of 

two different doses of NH. The low dose of NH produced 

2% inhibition of the enzyme system, whereas the high 

dose yielded 12% inhibition. 

Results 

Lead, ouabain, and NH were found to be potent 

inhibitors of the Na-K-ATPase enzyme system. The I50 

values for each substance are shown in Table 1. 

Kinetic Studies 

Potassium. The effects of varying concentrations 

of potassium on Na-K-ATPase activity in the presence 

of lead (5 x 10-5M), ouabain (4 x 10"9M), and NH (6.9 x 

IO"9 M) are shown in Figure 1. The inhibition of NaK-ATPase by ouabain was found to be competitive with 

respect to potassium, whereas the inhibitory effects of 

NH and lead were found to be noncompetitive. 

Sodium. With sodium as the variable sustrate, the 

kinetic analysis revealed that lead (2.5 x IO-5 M) is a 

competitive inhibitor (Fig. 2), whereas NH exhibited 

no inhibitory effect. 

Magnesium Adenosine Triphosphate 

(MgATP). With MgATP as the variable substrate, kinetic analysis revealed that lead is a competitive inhibitor; NH (1.3 x IO-7) exhibited uncompetitive inhibition (Fig. 3). The Km and Vmix values are listed in 

Tables 2 and 3. 

Synergy 

Figure 4 displays the dose-related inhibition of NaK-ATPase in the presence of increasing lead concentrations ranging from 0.1 x 10-" M to 5 x 10"4

 M. In the 

presence of low dose NH, the inhibition curve is shifted 

to the left, i.e., inhibition is increased. In the presence of 

high dose NH, a further leftward shift of the Na-KATPase inhibition curve was observed. In both instances, the increment of inhibition produced by addTable I. Determination of I50 values for inhibitors 

of Na-K-ATPase. 

Substance I(so.M' 

Ouabain 

Lead 

NH 

2X10-6 

80x10-* 

0.8 xlO-6 

aThe substrate concentrations for lead and ouabain are expressed a 

M, whereas the concentration for NH is expressed as M ouaba' 

equivalent. 
IF ""Vr

f

w

""r'

w

^iW1f""°'r "*-"" • 

P.4 

LEAD, NATRIURETIC HORMONE, AND NA-K-ATPASE 115 

:ng NH was more than would be predicted by a simple 

addition effect. 

2.4 -

2.0 • 

1.6 -

1/ V 1.2 • 

0.8 • 

0.4 f 

la / / • Enzyme 

T Ouabain 

o NH 

Pb 

8.0 

FIGURE 1. Lineweaver-Burk plot of kinetics of inhibition of Na-K-ATPase 

by lead (5 x IO"5 M), ouabain (4 x IO"9 M), and NH (0.69 x 10-9 M 

ouabain equivalents) with potassium as the variable substrate. 

Discussion 

The primary function of the membrane-bound NaK-ATPase enzyme system is the active transport of 

sodium and potassium (15,16). This enzyme system is 

also involved in salt and water homeostasis (17), the 

transport of nonelectrolytes (18), and the secretion of 

potassium and hydrogen by kidney tubules (18). The 

activity of the Na-K-ATPase enzyme system is affected 

by a variety of hormones, including mineralo- and 

glucocorticoids (19,20), thyroid hormone (21), 

insulin (22,23), catecholamines (24), and NH (25,26), 

as well as trace metals [e.g., lead, vanadium, mercury, 

uranium, cadmium, silver (8,9,27)]. This interaction 

c Pb 

o NH 

• Enzyme 

2.0 3.0 

1/[MgATP] 

FIGURE 3. Lineweaver-Burk plot of kinetics of inhibition of Na-K-ATPase 

by lead (5 x IO-5 M) and NH (1.3 x IO-7 M ouabain equivalents) with 

MgATP as the variable substrate. 

1.4 -

1.2 -

1.0 -

1/ V 0.8 • 

0.6 • 

0.4 -

0.2 - / *s"* 

/ i 

jS"^* 

m 

• Enzyme 

o pb 

-0.1 0.1 0.2 0.3 0.4 0.5 

l/(Na] 

FIGURE 2. Lineweaver-Burk plot of kinetics of inhibition of Na-K-ATPase by lead (25 x IO-6 M) with sodium as the variable substrate. 
P.5 

116 WEILER, KHAUL-MANESH, AND GONICK' 

Table 2 

Substrate 

Enzyme 

Lead 

NH 

Ouabain 

Table 3. 

Substrate 

Enzyme 

Lead 

NH 

Ouabain 

*m 

^ma> 

values for inhibitors of Na-K-ATPase. 

K 

0.48 

0.44 

1.61 

0.50 

tfm,mM 

Na 

34.9 

96.8 

MgATP 

2.69 

6.64 

values for inhibitors of Na-K-ATPase. 

v 

rmax 

K 

4.65 

3.48 

2.30 

4.77 

limole Pi/mg protein/hr 

Na 

17.54 

17.30 

MgATP 

20.16 

18.42 

10.13 

100 

- 80-

60-

o Pb + NH high dose 

• Pb + NH low dose 

cPb 

0.3 0.5 0.7 0.9 1.3 5.0 x 10 M 

Pb concentration 

FIGURE 4. Inhibition of Na-K-ATPase by lead in the presence and 

absence of low dose and high dose NH. The inhibitory effect of low 

dose NH alone was 2%; the inhibitory effect of high dose NH alone 

was 12% (plotted adjacent to y-axis). 

of metals with Na-K-ATPase may have physiological as 

well as toxicological significance. 

Na-K-ATPase has been classified as a sulfhydryl (SH) 

enzyme, in part because of its inhibition by mercurials (28). Lead, as a metal that reacts with SH groups, 

would also be predicted to act as a Na-K-ATPase inhibitor. Kramer et al, (9) found that in renal cortical 

tissue homogenates, Na-K-ATPase was inhibited by 

50% at a lead concentration of 7 x IO"5 M. Similar 

results were reported by Nechay and Saunders (7). 

These investigators showed that the concentration for 

50% inhibition of Na-K-ATPase ranged from 6 to 8 x 

IO-5 M in both microsomes and tissue homogenates. 

The homogenate did not contain any protective substances that ameliorated Na-K-ATPase inhibition, as 

had been reported for mercury (8). Both studies 

demonstrated that lead caused a reversible inhibition 

of the enzyme system. Ethylenediaminetetraacetic acid 

(EDTA) prevents or reverses lead-induced inhibition 

of Na-K-ATPase (7). Kramer et al. (9) demonstrated 

that lead is a competitive inhibitor with respect to its 

substrate ATP. From earlier studies by Tice (29), it 

would appear that lead competes with magnesium for 

chelation by ATP. Lead is also known to inhibit Na-KATPase at its sodium-sensitive site (7). In the present 

study, which employed a purified hog cerebral cortex 

Na-K-ATPase enzyme preparation, we also found that 

50% inhibition of the Na-K-ATPase enzyme system was 

achieved at a lead concentration of 8 x Iff5 M. The 

inhibition of Na-K-ATPase by lead was found to be 

noncompetitive with respect to potassium, but competitive with respect to sodium and MgATP. 

Endogenous inhibitors of Na-K-ATPase have been 

isolated from both plasma and urine by differing 

methodologies. Thus, the NH studied by various laboratories may consist of similar or different substances. Kramer et al. (30), studying a semi-purified 

urinary inhibitor, described the NH inhibition of NaK-ATPase as noncompetitive with respect to potassium. 

Crabos et al. (31) reported that NH, also isolated from 

urine, inhibited Na-K-ATPase noncompetitively with 

respect to ATP. Inhibition of the enzyme system was 

facilitated by sodium. Ouabain, a digitalis glycoside 

and a specific inhibitor of the Na-K-ATPase system, has 

been shown to be competitive with respect to potassium 

(32) and noncompetitive with respect to ATP (31). In 

the present study, we employed a semi-purified NH 

preparation derived from plasma of a patient with 

essential hypertension to perform kinetic studies. This 

NH inhibits Na-K-ATPase in a noncompetitive manner with respect to potassium and uncompetitively 

with respect to magnesium. The semi-purified material exerted no effect on the sodium site of the enzyme 

system. These results clearly demonstrate that NH and 

lead differ from each other and also from ouabain in 

their Na-K-ATPase inhibitory characteristics. 

At present, the role of lead as a contributory factor in 

hypertension remains speculative; however, there are 

in vitro data that support the possibility that lead enhances vascular reactivity. Webb et al. (33) found that 

tail arteries from hypertensive male rats exposed to lead 

at a dose of 100 ppm showed an increase in maximal 

contractile force when tested with the a-adrenergic 

agents, norephinephrine and methoxamine. This 

finding was attributed to an increase in the intracellular pool of activator calcium in the vascular 

smooth muscle cell. Piccini et al. (34) and Favalli et al. 

(35) found that calcium was necessary in the 

perfusion solution for lead to exert an effect on vascular 

contractility of the isolated rat tail artery. Furthermore, tissue calcium was increased in the presence of 

lead, an effect that could be explained by the inhibitory 

effect of lead on the vascular smooth muscle membrane 

Na-K-ATPase system, as predicted by the Blaustein 

hypothesis (11). As lead arid NH have a synergistic 

effect on this enzyme system, one possible action of lead 

may be to potentiate vascular reactivity in genetically 

susceptible individuals. Such a hypothesis

 lS 

supported by observations of Cloix et al. (36) that some 

normotensive individuals with a family history ° 

hypertension have elevated levels of NH. 

As hypertension is a multifactorial complex dis 

A 
LEAD, NATRIURETIC HORMONE, AND NA-K-ATPASE 117 

order, it is unlikely that a single hormone or toxin can 

account for the entire pathogenesis. A more plausible 

outcome is that many substances will be found to be 

contributory factors and the interplay between these 

substances will determine whether a given individual 

develops a sustained increase in blood pressure. 

The authors thank Ruby McCarty and Beverly Gil for their 

secretarial assistance. This study was supported by Endocrine 

Sciences Laboratories, the Louise and Gustavus Pfeiffer Foundation, 

and the International Lead Zinc Research Organization. 

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