BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 875-88]
Vol. 176, No. 2, 1991 April 30, 1991
STIMULATION OF ATRIAL NATRIURETIC PEPTIDE SECRETION AND SYNTHESIS BY NaK-ATPase INHIBITORS Toshio Morise, Yoshihiro Takeuchi, Shinya Okamoto and Ryoyu Takeda Second Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa City 920, Japan Received
March
18,
1991
Ouabain has been reported to increase the secretion of ANP in vitro. In this study, we focused on whether this action is common in Na-K-ATPase inhibitors (ATPI) and whether ATPI simply increase the release of ANP or stimulate both its biosynthesis and release. The effects of ouabain and digoxin on secretion of ANP and accumulation of ANP mRNAwere investigated in the rat cardiocyte superfusion system. Ouabain and digoxin increased the immunoreactive ANP(iANP) output into perfusate and accumulation of ANP mRNAsignificantly. These results suggest that ATPI may stimulate both ANP biosynthesis and r e l e a s e
in vitro.
© 1991 Academic Press, Inc.
Atrial natriuretic peptide(ANP), having various important biological activities,
including potent vasorelaxant(1) and natriuretic(2) properties,
a hormone produced mainly in the cardiac atria. interacts with other hormones. renin(3), aldosterone(4),
is
Interestingly, ANP also
It is known to suppress the secretion of
and vasopressin(5), and to reduce the bioactivity
of angiotensin(6) at the target organ level.
It is also known that ANP
secretion is stimulated by humoral factors, such as angiotensin If(9) and thyroid hormone(lO), as well as by stretching of the heart muscles(7,8). In addition,
i t has recently been reported that ouabain(ll), a cardiotonic
agent, has a stimulatory effect on ANP secretion,
This action of ouabain is
interesting when considering the cardiotonic mechanisms other than the positive
inotropic e f f e c t of t h i s drug,
however,
it
is s t i l l
uncertain
Abbreviation~: a t r i a l natriuretic peptide , ANP; Krebs-Ringer bicarbonate containing 0.1% glucose , KRBG:standard saline citrate, SCC; sodium dodecyl sulfate, SDS: Immunoreactive ANP , iANP. 0006-291X/91 $1.50 875
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
i) whether this effect is exclusive to ouabain or is common to Na-K-ATPase i n h i b i t o r s , and 2) whether the drug simply stimulates the release of ANP or stimulates both i t s the e f f e c t s of
biosynthesis and release.
ouabain and digoxin
To make these points c l e a r ,
which has also Na-K-ATPase inhibiting
property, on ANP secretion and the contents of ANP messenger RNA (mRNA) in the cardiocytes were studied.
!.
MATERIALS AND METHODS Preparation of rat myocardial suDerfusion preparation
Male Wistar rats weighing 250 g underwent thoracotomy under ether anesthesia, and their hearts were removed. The a t r i a were taken out, weighed, and cut into pieces 2 mm in size in the perfusion fluid(Krebs-Ringer bicarbonate containing 0.1% glucose: KRBG). Thesetissue pieces were placed in a p l a s t i c syringe 5 mm in diameter and 5 cm in length, which was covered with a piece of gauze folded into four at one end and with a rubber cap at the other end. Superfusion with oxygenated KRBGwas then performed at a flow rate of I ml/min. The perfusate was collected in a polypropylene tube and stored at -20"C until measurement of ANP levels. The perfusion experiment was performed using the a t r i a obtained from four rats. The a t r i a from four rats were cut into pieces, mixed well, and divided into seven equal portions. One portion was immediately treated for RNA extraction and used as the standard, while each of the remaining six portions was placed in a syringe for perfusion. One of them was perfused with KRBG only and used as the control, while the other five syringes were perfused with a perfusion solution containing, ouabain or digoxin (Sigma Co., Ltd., USA) at a concentration of i0 -o, 10-9 or ]O-5M. The a t r i a l t i s s u e pieces were f i r s t perfused with KRBGonly for 60 min, and then with either t e s t perfusate for a further 60 min. 2.RNA extraction RNA was extracted by the acid guanidium thiocyanate-pbenol-chloroform e x t r a c t i o n method(12). In b r i e f , a f t e r the completion of perfusion, the t i s s u e pieces were c o l l e c t e d and placed in 5 ml of denaturing solution (Solution D : 4M Guanidine Thiocyanate, 25mM Sodium Citrate pH4.0, 0.5% sarcocyl, 0.1M 2-mercaptoethanol). They were then homogenized with a g l a s s Teflon homogenizer, and subsequently t r a n s f e r r e d to a 10-ml polypropyrene tube. Subsequently, a mixture of 0.5 ml of 2 M sodium acetate (pH 4.0), 5 ml of saturated phenol, and i ml of chloroform-isoamyl alcohol were added to the homogenate. The suspension was shaken vigorously for 10 seconds, cooled on ice for 15 min, and then centrifuged at i0,000 g for 20 min at 4"C. After centrifugation, the aqueous layer was transferred to a new tube, mixed with 5 ml of i s opr o p an o l , and then kept at -90°C for I h to p r e c i p i t a t e RNA. Sedimentation was performed at 10,000 g for 20 min, and the r e s u l t a n t RNA p e l l e t was washed twice with 80% ethanol.
3.
Analysis of ANP mRNA
Total RNA was quantified by UV absorption at 260 nm. To measure the size of the hybridizing RNA band, Northern blot a n a l y s i s was performed. The denatured t o t a l RNA sample was size-fractionated on a 1.2% agarose gel c o n t a i n i n g 18.5% formaldehyde and t r a n s f e r r e d to a n i t r o c e l l u l o s e f i l t e r ( 1 3 ) . The f i l t e r was baked for 2 h at 80"C , and then prehybridized for 4 h at 42°C in a solution containing 50% formaldehyde, 5;: standard s a l i n e citrate(SCC), 25 ug denatured salmon sperm DNA, 0.1% sodium dodecyl sulfate (SDS), and ix Denhardt's solution. Hybridizationo~was performed for 12 h at 42"C in the same solution in the presence of a oZP-labeled FANPcDNA probe(25) which was kindly supplied by T, Uoguchi M.D., of Suntory Institute for Biomedical Research. The f i l t e r was washed in 0.3 x SCC containing 0.1% SDS at room temperature and autoradiographed for 24 h at -90*C. 876
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The level of ANP mRNA transcripts was determined by dot-hybridization technique of Berent et al. (26). For each sample, three serial dilutions of denatured t o t a l RNA were d o t t e d onto a n i t r o c e l l u l o s e f i l t e r . Baking, prehybridization, hybridization, and autoradiography were c a r r i e d out as described for the Northern blot analysis. Dot-blotting of the standard sample (RNAwas extracted immediately without perfusion) was performed simultaneously with processing of the samples from perfused a t r i a l tissue, and normalization was undertaken by considering the density of the standard sample as I. Relative ANP transcript levels were measured by densitometric scanning of the individual dots. 4, Measurement of immunoreaetive ANP Immunoreactive ANP(iANP) was measured by radioimmunoassay a f t e r s o l i d phase extraction using a SEP-PAK C18 cartridge (Millipore Corporation, Milford, MA, USA) as previously reported(14). The amount of iANP was expressed as that released into the fluid during 2hr of perfusion per I mg of the adrenal. RESULTS Effects of ouabain and di~oxin on ANP mRNA Northern blot hybridization showed a single band for a t r i a l RNA,
which
corresponded to the approximately 0.95 kb mRNAof ANP (Fig. I) and the amount of mRNAwas determined by dot-blotting(Fig. 2).
The content of ANP mRNA in
the a t r i a l muscle in the untreated group was 0.99 ± 0.07 and remained unchanged during 2 h of perfusion. with 10-6
The content showed no s i g n i f i c a n t changes
and lO-7 M ouabain in the perfusion fluid, but was significantly
elevated to 1.90 ± 0,17 (p