Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1990

Pages 514-520

ATRIAL NATRIURETIC PEPTIDE, NITROGLYCERINE, AND NITROPRUSSIDE REDUCE BASAL AND STIMULATED ENDOTHELIN PRODUCTION FROM CULTURED ENDOTHELIAL CELLS Outi

Saijonmaa 1.,

Ari Ristimfiki 2,

and

Frej Fyhrquist 1

1Unit of Clinical Physiology, Minerva Institute for Medical Research, Tukholmankatu 2 SF-00250 Helsinki, Finland and 2 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland Received October 26, 1990

SUMMARY Atrial natriuretic peptide (ANP) and the nitrovasodilator drugs nitroglycerine and nitroprusside were shown here to decrease both basal and thrombin stimulated production of endothelin-1 (ET-1) from cultured human endothelial cells as measured by radioimmunoassay. 8-Bromo-3',5'-cyclic guanosine monophosphate (cGMP) and papaverine also inhibited ET-1 production. The inhibitory effect of ANP and nitrovasodilators on ET-1 production thus appears to be mediated by guanylate cyclase and cGMP. Part of the vasodilatory action of ANP, nitroprusside and nitroglycerine may be due to suppression of endothelial ET-1 production. This may be an additional mechanism whereby nitrovasodilators participate in the regulation of vascular tone. ®~99oAoademicPress, ~o.

Vascular endothelium may play a critical role in the regulation of vascular tone by producing vasodilating and vasocontracting factors. Endothelin (ET), a 21-aminoacid peptide, first isolated from culture medium of porcine aortic endothelial cells, is the most potent vasoconstrictor known (1). Three distinct human endothelin related genes have been identified, coding for three peptides, ET-1, ET-2

and ET-3 (2).

Only ET-1 has been detected in endothelial cell culture medium (3). Various chemical and mechanical stimuli reportedly increase ET-1 production by cultured endothelial cells. These include thrombin (1), angiotensin II ( 4 ) , and fluid dynamical shear stress (5). Other factors reported to increase ET-1 concentration in endothelial cell culture are calcium

ionophores (1), E. coli endotoxin (6), insulin (7), and

To whom correspondence should be addressed. Abbreviations used in the text: ET-I, ET-2, ET-3,endothelinl,-2,-3; HPLC, high power liquid chromatography; cGMP, cyclic guanosine monophosphate; ANP, atrial natriuretic peptide. 0006-291X/90 $1. ~;0 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transforming growth factor-13 (8).

The mechanism of ET-1 production is unknown,

however. We here report inhibition human

of both basal and stimulated ET-1 production from

endothelial cells in culture by ANP and the exogenous nitrovasodilators,

nitroglycerine and nitroprusside.

MATERIALS AND METHODS Cell culture. Endothelial cells were prepared from human umbilical cord veins according to Jaffe et al. (9). Veins were cannulated, washed with phosphate buffered saline and treated with 0.5% collagenase (Sigma, St. Louis, MO,USA) in phosphate buffered saline for 15 min in 37oc water bath. Cells were grown to confluence on 0.2% gelatin (Sigma) coated cell culture flasks (Becton Dickinson, Switzerland) in Medium 199 (Gibco, UK) supplemented with 20 % fetal calf serum (Gibco), 20 Ixg/ml endothelial cell growth supplement (Sigma), 12 U/ml heparin (Sigma), 100 U/ml g-penicillin , 100 I.tg/ml streptomycin (Gibco) and 2 mM glutamine (Gibco) at 37 ° C in humidified 5% carbon dioxide in air. Medium was changed every other day. Cells were tested for viability with Trypan Blue. Experiments. Cells were detached with 0.125% trypsin- 0.02% versene solution (Gibco) and plated on 24 well plastic cell culture dishes (Nunc, Denmark) coated with 0.2% gelatin. The medium was changed every other day. At confluence the medium was replaced with Medium 199 containing 10% fetal calf serum and then incubated for 24 h. Before experiments the culture medium was aspirated from confluent monolayers and 1 ml of fresh Medium 199 with 5% fetal calf serum was added without additives (control) or containing following substances: nitroglycerine (Orion, Finland), 1-100 I.tg/ml, nitroprusside (Roche,BaseI,Schwitzerland), 1100 ixg/ml, atrial natriuretic peptide (human 1-28, Peninsula, London, UK), 0.1 10 ng/ml, 8-bromo- cyclic guanosine 3',5'- monophosphate (cGMP, Sigma), 50500 i.tg/ml,papaverine(Sigma), 101xg/ml, thrombin (Sigma), 2U/ml, or cycloheximide (Sigma), 0.45-45 i.tg/ml, and incubated for 4 h at 37°C in humidified 5% carbon dioxide in air. Culture medium was then stored at -20oc until measurement of ET immunoreactivity.

Endothelin radioimmunoassay. Radioimmunoassay of endothelin was performed as described (10), using synthetic ET-1 (Peptide Institute, UK) and ET antiserum generated in rabbits with ET-1 coupled by glutaraldehyde to keyhole limpet hemocyanin (Sigma) as an immunogen. The antiserum showed 100% cross-reaction with ET-2 and ET-3 (human; Peninsula,). Antiserum did not cr~ss-react significantly (

Atrial natriuretic peptide, nitroglycerine, and nitroprusside reduce basal and stimulated endothelin production from cultured endothelial cells.

Atrial natriuretic peptide (ANP) and the nitrovasodilator drugs nitroglycerine and nitroprusside were shown here to decrease both basal and thrombin s...
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