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The effects of salt-loading and dehydration on atrial natriuretic peptide (ANP) gene expression
Comp. Biochem. PhysioL Vol. 97B, No. I, pp. 205-208, 1990 Printed in Great Britain
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc
THE EFFECTS OF SALT-LOADING A N D D E H Y D R A T I O N ON ATRIAL NATRIURETIC PEPTIDE (ANP) GENE EXPRESSION MIN-HONG,* YAN JIN,* YIN-QIAO MAI* and KIA-KI HAN')':~ *Department of Biochemistry and Molecular Biology, Norman Bethune Medical University, ChangChun, Jilin Province 130.021, People's Republic of China; and ?Unit6 Inserm No. 16, Place de Verdun, 59.045 Lille Cedex, France (Tel: 0033 20 52 94 84) (Received 16 February 1990) Abstract--l. Effects of sodium loading and dehydration on ANP gene expression were investigated in rats. 2. ANP-mRNA was determined using Northern blot and dot blot hybridization technique with a-32p-labeled r-preproANP-cDNA probe. Salt loading increased the ANP-mRNA content in atria. Correlation with ANP-mRNA content and the plasma sodium concentration was established. 3. Deprivation of water for 2 and 4 days increased ANP-mRNA 2.1- and 1.6-fold, respectively. 4. These results demonstrated that water-salt balance affects the ANP-gene expression.
INTRODUCTION It has been well known for several years that mammalian cardiac atrium contains peptide hormones, atrial natriuretic peptides (ANP), with potent natriuretic, diuretic and vasorelaxant activities. Espiner and Richards (1989) and Hennth (1988) have reported the close related relationships in the regulation of sodium and fluid homeostasis. Because the plasma A N P levels does not fully reflect the dynamic equilibrium of A N P , especially concerning the A N P synthesis aspect, it will be interesting to share a light on the A N P gene expression. Although several investigators have recently reported that the changes in salt and water balance affect A N P levels in plasma or atria (Laragh et al., 1988), only limited information about the effects of water-salt balance on A N P gene expression have been published in the literature. Some data have shown that A N P effects rapid adjustments to large alterations in circulating fluid volume (Nguyen et al., 1988). Whereas Lattion et al. 0988) and Iwao (1988) independently reported the effects of sodium intake on ANP-gene expression during l to 3 weeks. However, little is known concerning the shorter-term effect of water-salt balance on A N P gene transcription. The present study was designed to investigate the effects of shorter-term salt-loading and dehydration on A N P gene expression in normal rat atrial myocardium. We also have dealt with the changing of plasma sodium and potassium concentrations during experimental periods. MATERIALS AND METHODS
Restriction enzymes Pstl, bovine serum albumin (BSA), tRNA, salmon sperm DNA (ss-DNA), nick translation kit were purchased from Boehringer Mannheim (FRG). Ficoll, polyvinyl pyrrolidone were provided from Sino-US :[:Author to whom correspondence should be addressed. 205
Biotechnology Company. a-32P-dATP was purchased from Amersham Chemical Company (UK). All the chemical reagents were of analytical grade. Most of the aqueous solutions were autoclaved or filtered by special filters (Millipore, USA). Bentonite was heated at 200°C for 3 hr before use. Plasmid pNI-11 was a gift from Professor Nicholas Barden. Animals Female Wistar rats weighing 220-250 g were nourished and maintained on regular pellet chow. The rats were divided into seven groups. Each group was constituted by six rats. Group 1 (Control) rats drank water. Groups 2, 3 and 4 (salt-loading) rats drank 0.9% NaCI for 2, 4 and 6 days, respectively. Whereas the groups 5, 6 and 7 (dehydration) rats were deprived of water for 2, 4 and 6 days, respectively. Animals were killed at the selected time. Under ether anesthesia, eye globes were taken out to collect blood. The atria was stored in liquid nitrogen (-195°C) until extraction or homogenized immediately. RNA extraction from tissues and criteria of purity Total RNA in atria was extracted with cold phenol methods as described by Griffin et al. (1978). Ten volumes of homogenization buffer (10 mmol sodium acetate pH 5.1, 0.1 mmol EDTA-2Na, 0.34% SDS and 0.5% bentonite), 5 vols of phenol (distilled and treated with homogenization buffer without bentonite, containing 0. ! % 8-hydroxyquinoline before use) and 5 vols of ehloroform-isoamylalcohol (v:v=24/1) were used per weight of tissues. The above mixtures were homogenized in an ice-bath, centrifuged at 4°C (10,000g) for 30rain. The supernatant fraction was then extracted with 0.5 vol of phenol, 0.5 vol of CHCI 3 once and then I vol of CHC 3, 0.1 vol of 1% dextra sulfate and 0.1 vol of 5% bentonite twice. Finally, the supernatant was added to 0.I vol of 3 mol sodium acetate pH 5.2 and 2.5 vol of cold absolute ethyl alcohol and stored together at -20°C overnight. After centrifugation, the RNA found in the pellet was washed for twice with 75% cold alcohol. The precipitated RNA was dried and then was resuspended in 1 x TE buffer (10mmol Tris-HCl pH8.0 containing 1 mmol of EDTA). RNA aliquotes were taken for the estimation of purity and content of RNA according to the ratio of spectrophotometric absorbance OD 260nm/280nm corresponding to I OD 260 = 40/zg/ml RNA.
MI~-HoNG et al.
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Northern blot analysis The procedures of Northern blot were performed under the conditions reported by Pfeiffer (1988). Sample preparations of 5pl RNA (10#g) was added to 20#1 sample denatured buffer (50% formamide, 2.2mmol formaldehyde, 25 mmol Hepes, 6 mmol sodium acetate pH 7.0 and 1 mmol EDTA 2Na) followed by heating at 60°C for 15min and then 10#1 sample loading buffer (20mmol PBS pH 6.8-7.2, 50% glycerol and 0.05% of Bromophenol Blue) was added. Northern blot filters were prepared by electrophoresis of RNA samples on a denaturing 0.8% agarose gel containing 20mmol PBS, pH 6.8-7.2, 2.2 mmol formaldehyde, 2 V/cm overnight at room temperature with subsequent transfer of the separated RNA onto a 0.45#m nitrocellulose filter by passive diffusion. Filters were then air-dried for 5 min and the RNA was fixed by baking at 80°C for 2 hr. Before transfer, the control sample gel was cut down and stained with ethidium bromide dye. Record of the electrophorectical migration distances of 28S and 18S RNA was then obtained by us.
2 ~
Dot blot analysis Serial dilutions of RNA in 10 × SSC ( x SSC = 155 mmol NaC1, 15 mmol sodium citrate pH 7.2) were spotted onto nitrocellulose filter. The RNA contents were 50, 25, 12.5, 6.25 and 3.125 #g/dot, respectively. Dot blot filter was dried in the same manner as Northern blot filter. Fig. 2. Northern blot assay electrophoresis migration distances. (1) 28S R N A = 5.7cm; (2) 18S RNA =7.8cm; (3) r-ANP-mRNA = 8.8 cm.
Preparation of r-preproANP-cDNA probe
U~
A ~
Plasmid pNI- 11 contains the r-preproANP-cDNA (r-ANP-cDNA) sequence with 650 bp approximately which was subcloned into PstI site of plasmid pGEM-2 (Hong et al., 1989). The ~x-32P labeled r-ANP-cDNA probe was prepared according to the following program 0.1 #g r-ANPeDNA, 3#1 dGTP, dCTP, dTTP mixtures (prepared by making a 1:1:1 mixture of solution of 0.4mmol/ldGTP, 0.4mmol/ldCTP, 0.4mmol/ldTTP), 2#1 of l0 x buffer, 2 #1 superior to 20 #Ci of (~- 32P) dATP and 3000 Ci/mmol aqueous solution, made up to 18 #1 with sterilized redistiUed water, 2 #1 of enzyme mixture (DNA polymerase-I, and DNAas¢-I in 50% glycerol). Incubation time was 35 min at 15°C. The reaction was stopped by adding 2/~1 of EDTA 0.2mmol/l, pH8.0. The non-incorporated deoxyribonucleoside tri-phosphates were removed by ethanol precipitation. Sodium acetate (1/10 vol of 3 mol) pH 5.2 and 2 vols of ethyl alcohol were added for 2 hr at - 50°C. The pellets were dissolved in an adequate amount of 1 x TE buffer.
Prehybridization, hybridization and washing The procedures used were similar to those used under experimental conditions reported by Pfeffer (1988). Nitrocellulose filters were prehybridized for 4 hr at 42°C in 50% formamide, 5 x SSC, 8 x Denhardt's (50 x Denhardt's = 50% ficoll, 5% polyvinyl pyrrolidone a n d 5% BSA), 50mmol sodium phosphate pH 6.5, 250#g/ml denatured salmon sperm DNA, 250 #g/ml tRNA and 0.1% SDS. The filters were then hybridized at 57°C for 16 hr in the same buffer containing 5 x 106 cpm of ~_32p labeled ANP-cDNA per 10 ml buffer. After hybridization, filters were washed for 20 min at room temperature 3 times in 1 x SSC 0.1% SDS, then washed at 65°C 1 hr and twice in 0.1 x SSC 0.1% SDS.
Fig. 1. RNA electrophoresis on denatured agarose. The gel was stained by Ethidium Bromide dye. (A) 18S RNA; (B) 28S RNA.
Autoradiography Finally filters were air-dried and exposed to Kodak X-ray film with two intensifying screens at -50°C for 7-10 days.
Effects of salt-loading
207
Table 1. The plasma sodium and potassium concentration (mmol/l) Control Days Groups Plasma (Na ÷ )
0.9% NaCI uptake 1 140.67 + 2.07
2 2 146.62 + 1.82"
4 3 149.83 + 2.79*
Plasma 4.45 4.46 4.13 (K ÷ ) +0.37 +0.32 +0.34 Data represents x + SD with *P < 5%.
Scan of hybridization signal The optical densities of the dot autoradiography were analyzed on a Laser densitometer. The statistical significance was analyzed as reported by Iwao (1988). Concentration and measurement of plasma (Na + ) and (K + ) The plasma samples were diluted 50-fold with redistilled water and then the concentration of plasma (Na ÷) and (K ÷) were automeasured with Corning 902 Flame photometer.
RESULTS AND DISCUSSIONS The ratio o f R N A samples was m o r e t h a n 1.8 R N A samples extracted from h e a r t atria were used in N o r t h e r n blot analysis. After electrophoresis on den a t u r e d f o r m a l d e h y d e agarose a n d e t h i d i u m b r o m i d e staining (see N o r t h e r n blot analysis), 28S a n d 18S R N A a p p e a r e d clearly u n d e r u.v. rays (Fig. 1). These results showed t h a t R N A samples are not degraded.
Water derivation 6 4 145.33 _+2.88*
2 5 148.17 + 2.56*
4 6 150.80 + 5.48*
6 7 155.67 + 3.39*
4.22 +0.47
3.80 +0.42
4.12 +_0.31
4.18 +_0.21
This control of n o n - d e g r a d e d R N A is necessary for further investigations. The resulting N o r t h e r n blot assay showed t h a t the hybridization signal o f r - p r e p r o A N P - m R N A a p p e a r e d at the front o f 18S R N A (Fig. 2). It was estimated a n d calculated to be near 8 0 0 - 1 0 0 0 b p length. This result was in agreement with those reported by T a k a y a n a g i et al. (1985) a n d by Seidman et al. (1984). In c o m p a r i s o n with the control group, salt-loading a n d d e h y d r a t i o n resulted in significant enhancem e n t of plasma (Na ÷) c o n c e n t r a t i o n whereas n o significant plasma (K ÷) c o n c e n t r a t i o n changes were observed (Table 1). D o t blot a u t o r a d i o g r a p h y a n d densities of d o t hybridization signals are s h o w n in Fig. 3 a n d Table 2, respectively. D o t blot analysis showed t h a t saltloading for 2, 4 a n d 6 days resulted in the significant increase o f levels o f r - A N P - m R N A content a n d the 2.4, 2.8 a n d 2.0-fold higher c o n t e n t t o w a r d the control group were o b t a i n e d by us. F u r t h e r m o r e ,
A
I
2
3
4
5
6
7
Fig. 3. The dot hybridization of rat atria RNA. (1) Control; (2), (3) and (4) were salt-loading groups for 2, 4 and 6 days, respectively; (5), (6) and (7) were dehydration groups for 2, 4 and 6 days, respectively. The total RNA content per dot: (A) 50 pg; (B) 25/~g; (C) 12.5/~g; (D) 6.25/~g; (E) 3.125/~g. Table 2. The densities of dot hybridization 0.9% NaC1 uptake Water deprivation 2 4 6 2 4 6 1 2 3 4 5 6 7 7.95 19.15 22.05 15.83 16.78 12.59 3.01 ___1.20 _+1.40" +0.41" +1.21" +2.10" -t-1.44" +0.34* Data represents x + SD with *P < 5%.
Control Days Groups Densities
208
MIN-HONG et al. CONCLUSION
300 o
The present experiments demonstrated that watersalt balance disorders could remarkably influence A N P gene expression. Both shorter term on more sodium intake and shorter term dehydration enhanced the A N P gene expression.
200
authors wish to express their thanks to Professor N. Barden for the valuable gift of plasmid pNI-I 1. This project was supported by a finanical subsidy of Chinese National Sciences Foundation to Professor Yin-Qiao Mai Furthermore, Professor Kia-Ki Han was supported by the United Nations' scholarship as an Expert of TOKTEN (Transfer of Knowledge Through Expatrial Nationals) from France to the People's Republic of China. Also, our thanks are due to the aids of French Foundation de Recherches Medicales de France. Acknowledgements--The
100
0 1
2
3
4
5
6
7
group
Fig. 4. The effects of salt-loading and dehydration on r-ANP-mRNA content in Atria. (1) Control; (2), (3) and (4) were salt-loading for 2, 4 and 6 days, respectively; (5), (6) and (7) were dehydration for 2, 4 and 6 days, respectively. the dehydration experiments revealed that if the deprivation of water lasted for 2 and 4 days, the r - A N P - m R N A content of those groups were enhanced more than 2.1- and 1.6-fold once compared to the control group. As the deprivation time of water was extended to 6 days, the relative content of r - A N P - m R N A in this group decreased significantly since it was only 0.38 times more than that of control group (Fig. 4). The results of these experiments revealed that much more sodium intake caused a significant enhancement of A N P gene expression. It speaks in favor that the dynamic changes of A N P - m R N A contents in atria were correlated and were in parallel with plasma sodium concentration but also the A N P m R N A content changed and inclined with respect to that of the control group during long sodium loading periods. Lattion et al. (1988) recently reported that after 1 week on a high sodium diet, A N P - m R N A was increased, whereas after 3 weeks the differences in cardiac A N P - m R N A levels had disappeared. Iwao (1988) reported that high sodium intake for 2 weeks did cause the slight elevation in the expression of the A N P gene. These experiments revealed that A N P was not responsible for long term regulation of sodium during sustained salt loading. Luft (1986) reported on the plasma A N P concentration change and their work was in good agreement with those reported both in the literature (Lattion et al., 1988; Iwao, 1988) and by our results. Concerning the influences of water deprivation on A N P gene expression, N a k a y a m a et al. (1984) and Zisfein (1986), showed that after water deprivation for 2, 4 and 5 days, the A N P - m R N A in atria were decreased. However we have shown that, at least, a shorter term of water deprivation could cause the enhancement of A N P gene expression.
REFERENCES
Espiner E. A. and Richards A. M. (1989) ANP--an important factor in sodium and blood pressure regulation. Lancet I April, 707-710. Griffin G. D., Sellin H. G. and Novelli D. (1978) Optimization of phenol extraction procedures for preparation of RNA from mammalian lymphoid organs. Analyt. Biochem. 87, 506-520. Hennth G. L. (1988) Physiology and pathophysiology of atrial peptide. Am. J. Physiol. 254, El-15. Hong M., Jin Y. and Mai Y. Q. (1989) Construction of "'in vitro" transcription system plasmid pNl-ll and pNI-12. J. Norman Bethune Univ. Med. Sci. 15, 433435. Iwao H. (1988) Sodium balance effects on rein angiotensinogen and A N P mRNA levels. Am. J. Physiol. 255 E129-136. Laragh J. H. et al. (1988) ANP: a regulator of blood pressure and volume homeostasis. Kidney Int. 34 (supplement 25), $64-71. Lattion A. L. et al. (1988) Effect of sodium intake on gene expression and plasma levels of ANP in rats. Am. J. Physiol. 255, H245-249. Luft F. C. (1986) ANP determinations and chronic sodium homeostasis. Kidney Int. 29, 1004-1010. Nakayama K., Ohkubo H., Hirose T., lnayama S. and Nakamishi S. (1984) mRNA sequence for human cardiodilatin ANP precursor and regulation of precursor mRNA in rat atria. Nature 310, 699-701. Nguyen P. V., Smith D. L. and Leenen F. H. H. (1988) Acute volume loading: ANP release and cardiac function in healthy men. Life Sci. 43, 821-830. Pfeiffer A. (1988) Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy. Molec. Cell. Endocr. 55, 115-120. Seidman C. E., Duby A. D., Choi E., Graham R. M., Haber E., Homcy C., Smith J. A. and Seidman J. G. (1984) The structure of rat preproatrial natriuretic factor as defined by a complementary DNA clone. Science 225, 324-326. Takayanagi R., Tanaka I., Maki M. and Inagami T. (1985) Effects of changes in water sodium balance of ANP messenger RNA and peptides in rats. Life Sci. 36, 1843-1848. Zisfein J. B. (1986) ANF: assessment of its structure in atria and regulation of its biosynthesis with volume depletion. J. Mol. Cell. Cardiol. 18, 917-929.
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc
THE EFFECTS OF SALT-LOADING A N D D E H Y D R A T I O N ON ATRIAL NATRIURETIC PEPTIDE (ANP) GENE EXPRESSION MIN-HONG,* YAN JIN,* YIN-QIAO MAI* and KIA-KI HAN')':~ *Department of Biochemistry and Molecular Biology, Norman Bethune Medical University, ChangChun, Jilin Province 130.021, People's Republic of China; and ?Unit6 Inserm No. 16, Place de Verdun, 59.045 Lille Cedex, France (Tel: 0033 20 52 94 84) (Received 16 February 1990) Abstract--l. Effects of sodium loading and dehydration on ANP gene expression were investigated in rats. 2. ANP-mRNA was determined using Northern blot and dot blot hybridization technique with a-32p-labeled r-preproANP-cDNA probe. Salt loading increased the ANP-mRNA content in atria. Correlation with ANP-mRNA content and the plasma sodium concentration was established. 3. Deprivation of water for 2 and 4 days increased ANP-mRNA 2.1- and 1.6-fold, respectively. 4. These results demonstrated that water-salt balance affects the ANP-gene expression.
INTRODUCTION It has been well known for several years that mammalian cardiac atrium contains peptide hormones, atrial natriuretic peptides (ANP), with potent natriuretic, diuretic and vasorelaxant activities. Espiner and Richards (1989) and Hennth (1988) have reported the close related relationships in the regulation of sodium and fluid homeostasis. Because the plasma A N P levels does not fully reflect the dynamic equilibrium of A N P , especially concerning the A N P synthesis aspect, it will be interesting to share a light on the A N P gene expression. Although several investigators have recently reported that the changes in salt and water balance affect A N P levels in plasma or atria (Laragh et al., 1988), only limited information about the effects of water-salt balance on A N P gene expression have been published in the literature. Some data have shown that A N P effects rapid adjustments to large alterations in circulating fluid volume (Nguyen et al., 1988). Whereas Lattion et al. 0988) and Iwao (1988) independently reported the effects of sodium intake on ANP-gene expression during l to 3 weeks. However, little is known concerning the shorter-term effect of water-salt balance on A N P gene transcription. The present study was designed to investigate the effects of shorter-term salt-loading and dehydration on A N P gene expression in normal rat atrial myocardium. We also have dealt with the changing of plasma sodium and potassium concentrations during experimental periods. MATERIALS AND METHODS
Restriction enzymes Pstl, bovine serum albumin (BSA), tRNA, salmon sperm DNA (ss-DNA), nick translation kit were purchased from Boehringer Mannheim (FRG). Ficoll, polyvinyl pyrrolidone were provided from Sino-US :[:Author to whom correspondence should be addressed. 205
Biotechnology Company. a-32P-dATP was purchased from Amersham Chemical Company (UK). All the chemical reagents were of analytical grade. Most of the aqueous solutions were autoclaved or filtered by special filters (Millipore, USA). Bentonite was heated at 200°C for 3 hr before use. Plasmid pNI-11 was a gift from Professor Nicholas Barden. Animals Female Wistar rats weighing 220-250 g were nourished and maintained on regular pellet chow. The rats were divided into seven groups. Each group was constituted by six rats. Group 1 (Control) rats drank water. Groups 2, 3 and 4 (salt-loading) rats drank 0.9% NaCI for 2, 4 and 6 days, respectively. Whereas the groups 5, 6 and 7 (dehydration) rats were deprived of water for 2, 4 and 6 days, respectively. Animals were killed at the selected time. Under ether anesthesia, eye globes were taken out to collect blood. The atria was stored in liquid nitrogen (-195°C) until extraction or homogenized immediately. RNA extraction from tissues and criteria of purity Total RNA in atria was extracted with cold phenol methods as described by Griffin et al. (1978). Ten volumes of homogenization buffer (10 mmol sodium acetate pH 5.1, 0.1 mmol EDTA-2Na, 0.34% SDS and 0.5% bentonite), 5 vols of phenol (distilled and treated with homogenization buffer without bentonite, containing 0. ! % 8-hydroxyquinoline before use) and 5 vols of ehloroform-isoamylalcohol (v:v=24/1) were used per weight of tissues. The above mixtures were homogenized in an ice-bath, centrifuged at 4°C (10,000g) for 30rain. The supernatant fraction was then extracted with 0.5 vol of phenol, 0.5 vol of CHCI 3 once and then I vol of CHC 3, 0.1 vol of 1% dextra sulfate and 0.1 vol of 5% bentonite twice. Finally, the supernatant was added to 0.I vol of 3 mol sodium acetate pH 5.2 and 2.5 vol of cold absolute ethyl alcohol and stored together at -20°C overnight. After centrifugation, the RNA found in the pellet was washed for twice with 75% cold alcohol. The precipitated RNA was dried and then was resuspended in 1 x TE buffer (10mmol Tris-HCl pH8.0 containing 1 mmol of EDTA). RNA aliquotes were taken for the estimation of purity and content of RNA according to the ratio of spectrophotometric absorbance OD 260nm/280nm corresponding to I OD 260 = 40/zg/ml RNA.
MI~-HoNG et al.
206
Northern blot analysis The procedures of Northern blot were performed under the conditions reported by Pfeiffer (1988). Sample preparations of 5pl RNA (10#g) was added to 20#1 sample denatured buffer (50% formamide, 2.2mmol formaldehyde, 25 mmol Hepes, 6 mmol sodium acetate pH 7.0 and 1 mmol EDTA 2Na) followed by heating at 60°C for 15min and then 10#1 sample loading buffer (20mmol PBS pH 6.8-7.2, 50% glycerol and 0.05% of Bromophenol Blue) was added. Northern blot filters were prepared by electrophoresis of RNA samples on a denaturing 0.8% agarose gel containing 20mmol PBS, pH 6.8-7.2, 2.2 mmol formaldehyde, 2 V/cm overnight at room temperature with subsequent transfer of the separated RNA onto a 0.45#m nitrocellulose filter by passive diffusion. Filters were then air-dried for 5 min and the RNA was fixed by baking at 80°C for 2 hr. Before transfer, the control sample gel was cut down and stained with ethidium bromide dye. Record of the electrophorectical migration distances of 28S and 18S RNA was then obtained by us.
2 ~
Dot blot analysis Serial dilutions of RNA in 10 × SSC ( x SSC = 155 mmol NaC1, 15 mmol sodium citrate pH 7.2) were spotted onto nitrocellulose filter. The RNA contents were 50, 25, 12.5, 6.25 and 3.125 #g/dot, respectively. Dot blot filter was dried in the same manner as Northern blot filter. Fig. 2. Northern blot assay electrophoresis migration distances. (1) 28S R N A = 5.7cm; (2) 18S RNA =7.8cm; (3) r-ANP-mRNA = 8.8 cm.
Preparation of r-preproANP-cDNA probe
U~
A ~
Plasmid pNI- 11 contains the r-preproANP-cDNA (r-ANP-cDNA) sequence with 650 bp approximately which was subcloned into PstI site of plasmid pGEM-2 (Hong et al., 1989). The ~x-32P labeled r-ANP-cDNA probe was prepared according to the following program 0.1 #g r-ANPeDNA, 3#1 dGTP, dCTP, dTTP mixtures (prepared by making a 1:1:1 mixture of solution of 0.4mmol/ldGTP, 0.4mmol/ldCTP, 0.4mmol/ldTTP), 2#1 of l0 x buffer, 2 #1 superior to 20 #Ci of (~- 32P) dATP and 3000 Ci/mmol aqueous solution, made up to 18 #1 with sterilized redistiUed water, 2 #1 of enzyme mixture (DNA polymerase-I, and DNAas¢-I in 50% glycerol). Incubation time was 35 min at 15°C. The reaction was stopped by adding 2/~1 of EDTA 0.2mmol/l, pH8.0. The non-incorporated deoxyribonucleoside tri-phosphates were removed by ethanol precipitation. Sodium acetate (1/10 vol of 3 mol) pH 5.2 and 2 vols of ethyl alcohol were added for 2 hr at - 50°C. The pellets were dissolved in an adequate amount of 1 x TE buffer.
Prehybridization, hybridization and washing The procedures used were similar to those used under experimental conditions reported by Pfeffer (1988). Nitrocellulose filters were prehybridized for 4 hr at 42°C in 50% formamide, 5 x SSC, 8 x Denhardt's (50 x Denhardt's = 50% ficoll, 5% polyvinyl pyrrolidone a n d 5% BSA), 50mmol sodium phosphate pH 6.5, 250#g/ml denatured salmon sperm DNA, 250 #g/ml tRNA and 0.1% SDS. The filters were then hybridized at 57°C for 16 hr in the same buffer containing 5 x 106 cpm of ~_32p labeled ANP-cDNA per 10 ml buffer. After hybridization, filters were washed for 20 min at room temperature 3 times in 1 x SSC 0.1% SDS, then washed at 65°C 1 hr and twice in 0.1 x SSC 0.1% SDS.
Fig. 1. RNA electrophoresis on denatured agarose. The gel was stained by Ethidium Bromide dye. (A) 18S RNA; (B) 28S RNA.
Autoradiography Finally filters were air-dried and exposed to Kodak X-ray film with two intensifying screens at -50°C for 7-10 days.
Effects of salt-loading
207
Table 1. The plasma sodium and potassium concentration (mmol/l) Control Days Groups Plasma (Na ÷ )
0.9% NaCI uptake 1 140.67 + 2.07
2 2 146.62 + 1.82"
4 3 149.83 + 2.79*
Plasma 4.45 4.46 4.13 (K ÷ ) +0.37 +0.32 +0.34 Data represents x + SD with *P < 5%.
Scan of hybridization signal The optical densities of the dot autoradiography were analyzed on a Laser densitometer. The statistical significance was analyzed as reported by Iwao (1988). Concentration and measurement of plasma (Na + ) and (K + ) The plasma samples were diluted 50-fold with redistilled water and then the concentration of plasma (Na ÷) and (K ÷) were automeasured with Corning 902 Flame photometer.
RESULTS AND DISCUSSIONS The ratio o f R N A samples was m o r e t h a n 1.8 R N A samples extracted from h e a r t atria were used in N o r t h e r n blot analysis. After electrophoresis on den a t u r e d f o r m a l d e h y d e agarose a n d e t h i d i u m b r o m i d e staining (see N o r t h e r n blot analysis), 28S a n d 18S R N A a p p e a r e d clearly u n d e r u.v. rays (Fig. 1). These results showed t h a t R N A samples are not degraded.
Water derivation 6 4 145.33 _+2.88*
2 5 148.17 + 2.56*
4 6 150.80 + 5.48*
6 7 155.67 + 3.39*
4.22 +0.47
3.80 +0.42
4.12 +_0.31
4.18 +_0.21
This control of n o n - d e g r a d e d R N A is necessary for further investigations. The resulting N o r t h e r n blot assay showed t h a t the hybridization signal o f r - p r e p r o A N P - m R N A a p p e a r e d at the front o f 18S R N A (Fig. 2). It was estimated a n d calculated to be near 8 0 0 - 1 0 0 0 b p length. This result was in agreement with those reported by T a k a y a n a g i et al. (1985) a n d by Seidman et al. (1984). In c o m p a r i s o n with the control group, salt-loading a n d d e h y d r a t i o n resulted in significant enhancem e n t of plasma (Na ÷) c o n c e n t r a t i o n whereas n o significant plasma (K ÷) c o n c e n t r a t i o n changes were observed (Table 1). D o t blot a u t o r a d i o g r a p h y a n d densities of d o t hybridization signals are s h o w n in Fig. 3 a n d Table 2, respectively. D o t blot analysis showed t h a t saltloading for 2, 4 a n d 6 days resulted in the significant increase o f levels o f r - A N P - m R N A content a n d the 2.4, 2.8 a n d 2.0-fold higher c o n t e n t t o w a r d the control group were o b t a i n e d by us. F u r t h e r m o r e ,
A
I
2
3
4
5
6
7
Fig. 3. The dot hybridization of rat atria RNA. (1) Control; (2), (3) and (4) were salt-loading groups for 2, 4 and 6 days, respectively; (5), (6) and (7) were dehydration groups for 2, 4 and 6 days, respectively. The total RNA content per dot: (A) 50 pg; (B) 25/~g; (C) 12.5/~g; (D) 6.25/~g; (E) 3.125/~g. Table 2. The densities of dot hybridization 0.9% NaC1 uptake Water deprivation 2 4 6 2 4 6 1 2 3 4 5 6 7 7.95 19.15 22.05 15.83 16.78 12.59 3.01 ___1.20 _+1.40" +0.41" +1.21" +2.10" -t-1.44" +0.34* Data represents x + SD with *P < 5%.
Control Days Groups Densities
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MIN-HONG et al. CONCLUSION
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The present experiments demonstrated that watersalt balance disorders could remarkably influence A N P gene expression. Both shorter term on more sodium intake and shorter term dehydration enhanced the A N P gene expression.
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authors wish to express their thanks to Professor N. Barden for the valuable gift of plasmid pNI-I 1. This project was supported by a finanical subsidy of Chinese National Sciences Foundation to Professor Yin-Qiao Mai Furthermore, Professor Kia-Ki Han was supported by the United Nations' scholarship as an Expert of TOKTEN (Transfer of Knowledge Through Expatrial Nationals) from France to the People's Republic of China. Also, our thanks are due to the aids of French Foundation de Recherches Medicales de France. Acknowledgements--The
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Fig. 4. The effects of salt-loading and dehydration on r-ANP-mRNA content in Atria. (1) Control; (2), (3) and (4) were salt-loading for 2, 4 and 6 days, respectively; (5), (6) and (7) were dehydration for 2, 4 and 6 days, respectively. the dehydration experiments revealed that if the deprivation of water lasted for 2 and 4 days, the r - A N P - m R N A content of those groups were enhanced more than 2.1- and 1.6-fold once compared to the control group. As the deprivation time of water was extended to 6 days, the relative content of r - A N P - m R N A in this group decreased significantly since it was only 0.38 times more than that of control group (Fig. 4). The results of these experiments revealed that much more sodium intake caused a significant enhancement of A N P gene expression. It speaks in favor that the dynamic changes of A N P - m R N A contents in atria were correlated and were in parallel with plasma sodium concentration but also the A N P m R N A content changed and inclined with respect to that of the control group during long sodium loading periods. Lattion et al. (1988) recently reported that after 1 week on a high sodium diet, A N P - m R N A was increased, whereas after 3 weeks the differences in cardiac A N P - m R N A levels had disappeared. Iwao (1988) reported that high sodium intake for 2 weeks did cause the slight elevation in the expression of the A N P gene. These experiments revealed that A N P was not responsible for long term regulation of sodium during sustained salt loading. Luft (1986) reported on the plasma A N P concentration change and their work was in good agreement with those reported both in the literature (Lattion et al., 1988; Iwao, 1988) and by our results. Concerning the influences of water deprivation on A N P gene expression, N a k a y a m a et al. (1984) and Zisfein (1986), showed that after water deprivation for 2, 4 and 5 days, the A N P - m R N A in atria were decreased. However we have shown that, at least, a shorter term of water deprivation could cause the enhancement of A N P gene expression.
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