Stimulation of Na-K-Cl cotransport in cultured vascular endothelial cells by atrial natriuretic peptide

Vol. 159, No. 2, 1989 March 15, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 734-740

STIMULATION OF Na-K-Cl COTRANSPORT IN CULTURED VASCULAR ENDOTHELIAL CELLS BY ATRIAL NATRIURETIC PEPTIDE Tetsuro

Fujita,

Department

Received

Hiromi Masami

of Biological Ookayama, January

30,

Hagiwara, Shunji Ohuchi, Masamichi and Shigehisa Hirose Ishido, Sciences, Meguroku,

Tokyo Institute Tokyo 152, Japan

of

Kozuka,

Technology,

1989

Vascular endothelial cells have been shown to contain atria1 natriuretic peptide (ANP)-sensitive Na-K-Cl cotransport system whose activity is regulated by intracellular FGMP levels. Addition of ANP to culture medium stimulated seRb uptake in bovine endothelial cells with a concomitant increase in cGMP contents. This action of ANP was mimicked by 8-bromo-cGMP and These results indicate that completely diminished by furosemide. ANP selectively activates the Na-K-Cl cotransporter in vascular endothelial cells via cGMP and offer new insight into the 0 1989 physiological significance of endothelial ANP receptors. Academic

Press,

Inc.

Specific receptors for atria1 natriuretic peptide (ANP) have been identified in a variety of tissues involved in the regula__ tion of blood pressure and fluid homeostasis (1,2). Most of their locations are well understood with respect to their physiological roles (3-5); for example, the receptors on the vascular smooth muscle cells, epithelial cells of the kidney, and zona glomerulosa cells of the adrenal are known to mediate the vasorelaxant, natriuretic, and aldosterone secretion-inhibitory actions of ANP, respectively (6-12). In addition to these sites where the physiological significance of ANP has been established, the presence of ANP receptors has also been demonstrated in tissues and cells where the function of ANP is not known. These include the endothelial cells in which we were interested since earlier work using cultured vascular endothelial cells has demonstrated that ANP causes intracellular accumulation of cGMP, suggesting that ANP may play some important roles in the regulation of the endothelial cell functions (8,13). Since ANP acts in the direction of reducing extracellular fluid volume in most target tissues, we speculated that the 0006-291X/89 $1.50 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

734

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the ion permeability of the enatria1 peptide may influence To evaluate this hypothesis, we examined the dothelial cells. effects of ANP on the ion transport systems in the endothelial cells and found that ANP increases the Na-K-Cl cotransporter activity by modulating the intracellular cGMP levels. Na-K-Cl cotransporter is a transmembrane protein present in many animal cell types and catalyzes the translocation of Na+, and ClThe with a stoichiometry of 1:1:2 (14,15). K+ , cotransporter is sensitive to loop diuretics such as furosemide and known to bind Rb+ to K+-binding site with equal efits ficiency (16). In the present study, we demonstate the presence of furosemide-sensitive Na-K-Cl cotransport system in the bovine endothelial cells using 86Rb+ as the tracer for K* influx and show that its activity is regulated by ANP. MATERIALS

AND METHODS

Materials--Fresh bovine carotid artery was obtained from a local slaughterhouse. Fetal bovine serum and cell culture medium (Eagle's minimum essential medium) were from Gibco; a6RbCl was from Du Pont-New England Nuclear; rat ANP was from Peptide Institute; ouabain, furosemide, 8-bromo-cGMP, 8-bromo-CAMP, and isobutylmethylxanthine(IBMX) were from Sigma; radioimmunoassay kit for cGMP was from Yamasa. Cells--Endothelial cells were prepared from a bovine carotid as previously described (17). The cells were maintained Eagle's minimum essential medium (MEM) containing 10% fetal itvine serum (FBS), 50 units/ml of penicillin and 50 ug/ml of streptomycin at 37OC in a 5% CO2 humidified incubator. All cells were used between the 6th and 21st passages. artery

Ion Transport Measurement--Prior to ion transport measurement, cells+were subcultured in petridishes (55 cm:) with culture medium. K influx was assessed by measuring s6Rb *uptake, since Rb+ was found to substitute quantitatively for K in Na-K-Cl cotransport (16). The cells (lOe/dish) were washed with serumfree MEM, and incubated for 15 min at 37OC with the medium containing 1 mM ouabain, 100 uM furosemide, or various reagents to be tested. This medium was aspirated and replaced with fresh MEM +containing 0.1 uM ANP. After a 25-min incubation, 1 uCi/ml seRb was added to the medium and cultures were incubated for an additional 5 min at 37OC. Then the cells were thoroughly washed to remove extra isotopes, and solubilized with 2 ml of 0.5 N NaOH. The alkaline extracts were countyd in a liquid scintillation counter in order to quantitate s6Rb influx. equiIntracellular cGMP Measurement--Endothelial cells After librately subcultured in petridishes (55 cmz) were used. washing the cells with serum-free MEM, various concentrations of rat ANP were added to the medium and the cells were incubated for The medium was aspirated and 2 ml of 6 % 30 min at 37oc. to the dishes. The cellular trichloroacetic acid was added extracts were centrifuged for 10 min at 1,600 x g and the super735

Vol. 159, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

natant fractions were extracted with ether to water-saturated remove trichloroacetic acid. The amounts of cGMP in the supernatants were quantified by radioimmunoassay using a commercially available kit. RESULTS ANP-sensitive Na-K-Cl Contransport in Endothelial Cells-Vascular endothelial cells from bovine carotid artery were cultured in MEM and their ability to translocate monovalent ions across the plasma membrane was examined using =Rb+. As shown in Fig. 1, the cells exhibited time-dependent uptake of 86Rb+. 86 Based on this kinetic data, Rb+ uptake was measured at 5 min in the subsequent experiments designed to examine the effect of ANP. Addition of ANP increased the 86Rb+ influx (Fig. 1). Fig. 2 shows the dose-response relationship between the concentration of ANP and stimulation of 86 Rb+ uptake. We next attempted to determine which ion transport system is sensitive to ANP using various inhibitors (Fig. 3); ouabain, a specific inhibitor of Na+, K+ATPase, substantially decreased the 86Rb+ influx but did not affect the ANP-induced increment of 86Rb+ uptake: however, furosemide, an inhibitor of Na-K-Cl cotransporter, diminished the stimulatory effect of ANP. These results indicate that ANP stimulates exclusively the Na-K-Cl cotransporter activity among other monovalent ion transporters in the endothelial cells.

2

4 Time

6

8

02

11

10 - log 9 M 8

7

6

(mlnl

Fig. 1. Time course of 8sRb+ uptake in endothelial uptake was assayed as described under "Materials the absence (0) or presence (0) of 0.1 uM ANP.

cells. 88Rb+ and Methods" in

Fig. 2. Dose-response curve of ANP stimulation of 88Rb* influx and cGMP accumulation in endothelial cells. 86Rb+ influx ( 0 ) and cGMP accumulation ( l ) were assayed as described under "Materials and Methods". The phosphodiesterase inhibitor IBEX (1 mM) was added to all samples and incubated for 5 min at 37OC prior to assay. 736

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Vol. 159, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

Ouabain rwoseinlae

-

-

ANP

-

+

++ --+

-++ -+

+ + ++ -+

Fig. 3. Effects of yarious ion transport inhibitors on basal and ANP-stimulated asRb uptake. Cells were preincubated for 15 min in serum-free MEM containipg 1 mM IBMK and 1 mMouabain or 100 uM furosemide, and their seRb transport activities were measured as described under "Materials and Methods". Each bar represents means f S.D. of 4-5 experiments.

Cyclic GMP as the reported by Schenk -et al -* tured vascular endothelial in part,

the

ANP-stimulated

Na-K-Cl 86Rb+

cGMP levels.

We

86Rb+

and the

uptake

and cGMP level

cells

were

Regulation

treated

cells.

If

cGMP levels

cotransporter

level

at

least

the

magnitude

with

intracellular

examined

the

relationship

cellular

increased with

govern,

correlate

of

activity,

previously in cul-

should

uptake

therefore

uptake

Mediator of ANP Action--As (81, ANP elevated cGMP levels

cGMP (Fig.

coordinately

increasing

between

2);

when

amounts

of

both

the

86Rb+

cultured

of ANP. Furthermore,

Table I of Na-K-Cl cotransport system in endothelial cGMP

cells

by

86Rb+ uptake (nmol/min/mg protein)

Addition

None IBMK (1 mM) ANP (0.1 UM) 8-bromo-cGMP (100 uM) 8-bromo-cGMP and ANP 8-bromo-CAMP (100 uM)

1.77 1.80 2.47 2.30 2.55 1.73

f f f f f f

0.18 0.07 0.30 0.09 0.07 0.06

Cells were preincubated for 5 min in serum-free MEM containing 1 mM IBMK and then treated with 0.1 UM ANP, 100 uM 8bromo-cGMP. or 8-bromo-CAMP for 30 min at 37OC. seRb+ uptake assay was carried out as described under "Materials and Methods". Values are means f S.D. of 3-5 experiments. 737

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the effect of ANP could be mimicked by 8-bromo-cGMP, a membranepermeable analog of cGMP (Table I). The stimulatory effects of ANP and 8-bromo-cGMP were not additive. In contrast to the cGMP analog, the permeable CAMP analog 8-bromo-CAMP did not affect the 86Rb+ uptake. These results indicate that the ANP-induced stimulation of Na-K-Cl cotransport is mediated via increase in intracellular cGMP levels.

DISCUSSION Animal

cells have many ion transport systems to maintain a suitable internal ionic environment or to actively participate in Especially, osmoregulation and solute secretion and absorption. monovalent ion transport systems such as Na+, K+-ATPase (18), Naantiporter (19.20), Ca'+K-Cl cotransporter (14,151, Na+/H+ play major roles in dependent K+ channel (21-24) and Cl- channel cellular volume regulation and in the maintenance of normal body salt and fluid. These systems have mainly been studied using intestine (26,27); the epithelial cells of the kidney (25) and vascular cells have not been examined systems in endothelial intensively. Our original interest in vascular endothelial cells lay in the fact that the cells have abundant ANP receptors of unknown physiological function. In the present study, therefore, we began to probe for the physiological action of ANP in the cultured bovine carotid artery endothelial cells by examining if ANP influences the transport processes of monovalent cations. The data obtained establish that ANP selectively activates Na-K-Cl cotransport in the endothelial cells. Na-K-Cl cotransport systems in other cell types have also been shown to be under the control of ANP: O'Grady et al . (27) have reported that ANP inhibits the Na-K-Cl cotransporter in the intestinal mucosa of a marine teleost; O'Donnel and Owen (28-30) have demonstrated the existence of ANP-stimulatable Na-K-Cl cotransporter in vascular smooth muscle cells and suggested its possible link to the stimulus-transfer pathway by which ANP causes vasorelaxation. The stimulatory effect of ANP on the vascular endothelial cotransporter (present study) and the opposite inhibitory effect observed in intestinal epithelial cells (27) appear to be quite reasonable if it is taken into account that the general function of ANP is to reduce extracellular fluid volume. 738

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ACKNOWLEDGMENTS This work was supported by grants-in-aid Research from the Ministry of Education, Japan, The Ito Memorial Foundation, Chichibu International Scientific Research Program. Nagata for her secretarial assistance.

for Scientific Science and Culture, Cement, and Mombusho We thank Yasuko

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