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The decline of atrial natriuretic peptide (ANP) gene expression in older rats and the effects of ginsenoside on ANP gene expression
Comp. Biochem. Physiol. eel. 101B,No. 1/2, pp. 35-39, 1992 Printed in Great Britain
0305-0491/92$5.00+ 0.00 © 1992PergamonPresspig
THE DECLINE OF ATRIAL NATRIURETIC PEPTIDE (ANP) GENE EXPRESSION IN OLDER RATS A N D THE EFFECTS OF GINSENOSIDE ON ANP GENE EXPRESSION MING HONG,* YAN JIN,* YIN-QIAO MAI,* ARNOLD BOERSMA,?KIA-KI HAN,t~ MARIE-CHRISTINEVANTYGI-IEM§and JEAN LEWBVRE§ *Department of Biochemistry and Molecular Biology Laboratory, Norman Bethune University of Medical Sciences,Chang-Chun, Jilin Province, 130.021,People's Republic of China; ~'Unit6INSERM No. 16, Place de Verdun, 59045, Lille C6dex, France (Tel: 0033-20-52. 94.84; (Fax: 0033-20-52. 70.83); and §Departement d'Endocrinologie et M6tabolismes, USN-A, Rue du Professenr Laguesse, 59.037, Lille C6dex, France
(Received 5 July 1991) Abstract--1. The levels of atrial natriuretic peptide(ANP) gone expression in rat atria at 2-3 and 14-18 months of age and the effects of ginsenosideson r-ANP gone expression by determining the concentration of ANP-mRNA were investigated. The male and femalerats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with a-32*P-labelled r-prepro-ANP-cDNA probe. 2. The ANP-mRNA contents of 14-18 month rats were remarkably less than that of 2-3 month rats. The levels of male and female rats' atria at 14-18 months were about 15 and 60%, the content of male and female rats at 2-3 months respectively. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14-18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2-3 months. 4. These results revealed that the ANP gone expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.
INTRODUCTION Over the past decade, an important hormone has been discovered and studied in detail atrial natriuretic peptide (ANP) or named atrial natriuretic hormone. ANP is a polypeptide hormone produced by atrial heart muscle. It is a potent diuretic (natriuretic) and hypotensive agent (Espiner and Richards, 1989; Hennth, 1988). ANP is a single copy gene, which is located on the distal short arm of a human chromosome (Yang-Feng et al., 1985). The rat A N P - m R N A is nearly 800-1000 bases (Seidman e t a l . , 1984; Nakayama etal., 1984) and it is translated to give rise to a prepro-ANP. Cloning and sequence analysis of eDNA for ANP precursor show that ANP is synthesized in a prepro form containing 152 amino acids (rat) or 151 amino acids (human) (Oikawa et al., 1985). A disulphide-looped sequence of 17 amino acids with various C- and N-terminal extensions is necessary for activity. In rat and human atrial granules and homogenate, the main form of ANP is a 126-residue peptid¢ called cardionatrin IV or ),-ANP;
~/Author to whom correspondence should be addressed. Abbreviations used--ANP, atrial natriuretic peptide or atrial natriuretic hormone; G-PSL, ginsenosides prepared from ginseng stems and leaves; G-PR, ginsenosides, prepared from ginseng roots. 35
this is derived from prepro-ANP by processing. Circulating ANP appears to be a 28-residue peptide, called cardionatrin-I or ~-ANP. Both human ~-ANP and rat ~-ANP are constituted by 28 amino acid residues (Lewicki et al., 1986). The ontogenic development regulation of ANP gone expression has been thoroughly investigated in rats. In utero, both atria and ventricules produce ANP. In the newborn rats, the A N P - m R N A in atria was not detected or its level was very low, but after birth the ANP gone expression was soon activated. The activation has been demonstrated by the progressive accumulation of ANP-mRNA; simultaneously the ANP concentration in the adult atria increases 6-fold relative to foetal atria. In contrast, with ANP gone expression enhancement in atria, the levels of ventricular A N P - m R N A gradually decreased. The ventricular A N P - m R N A in adults was only 1% of that in neonatal rat (Bloch et al., 1986; Wei et al., 1987; Wu etal., 1988). Little was known about the state of ANP gene expression during ageing development. Ginseng is a classical Chinese herb, which has been used in medicine for more than 1000 years. It is therefore interesting to know whether ginseng can influence the endocrinological function of the heart or not. In this paper, we deal with the decline of A N P - m R N A levels during ontogenic ageing devel-
36
MING HONG et al.
o p m e n t a n d the effects o f ginsenoside o n A N P gene expression.
MATERIALS AND METHODS
A Nick translation kit was purchased from Boehringer Mannheim (Germany), bovine serum albumin (BSA), salmon sperm DNA (ss-DNA), restriction enzyme PstI, Ficoll, and polyvinyl pyrrolidone were purchased from the Sino-US Biotechnology Company in Beijing (China) and from the Sigma Chemical Co. (St Louis, MO). All the chemical reagents usedin these experiments were analytical grade. Most of the aqueous solutions were autoclaved or filtered by special microhole filters (Pharmacia, Sweden). Bentonite was heated at 200°C for 3 hr before use. Plasmic pNI-ll was a gift from Prof. Niocholas Barden. Ginsenosides prepared from ginseng stems and leaves (G-PSL) and from ginseng roots (G-PR) were provided by Prof. Xiang Zhou as a gift.
Animals Both male and female rats of different ages (in months) were used. They were nourished and maintained on a regular pellet diet and tap water. The male Wistar rats were 2-3 and 14-18 months in age with body wts of 200 + 20 and 380 __+40 g, respectively. Female Wistar rats at 2-3 and 14-18 months of 220 __+350 and 350 ___30 g were used for the experiment. The male rats were divided into six groups, which include groups 1-3 (2-3 months) and groups 4-6 (14-18 months). Each group had 8-10 rats. Group 1 and group 4 rats were abdominally (i.p.) injected with a suitable volume of distilled water as the control group. Group 2 and group 5 rats were i.p. injected with aqueous solution of 1% of G-PSL (50 mg/kg body wt once a day for 7 days serially) (G-PSL groups). Group 3 and group 6 rats were i.p. injected with aqueous solution of 1% G-PR once a day for 7 consecutive days (G-PR groups); the dose was the same as that for G-PSL groups. The contents of the female rat groups were identical to the male groups as described above.
Preparation of RNA samples and criteria of purity and quantity. Animals were killed at the selected time. Under ether anaesthesia, the eye globes of rats were taken out to collect blood. The atria were removed after decapitation as quickly as possible and stored in liquid nitrogen ( - 195°C) until RNA extraction, or homogenized immediately. The total RNA in atria was extracted with cold phenol methods as described by Griffin et al. (1987). Ten volumes of homogenization buffer (10 mmol sodium acetate pH 5.1; 0.1 mmol EDTA-2Na; 0.34% of SDS and 0.5% bentonite), 5 vols of phenol (which was redistilled and treated with homogenization buffer without bentonite, containing 0.1% 8-hydroxyquinoline before use) and 5 vols of chloroformisoamylalcohol (24:1, v/v) were used per weight of tissues. The above mixtures were homogenized in an ice bath and centrifuged at 4°C (10,000 g) 30 min. The supernatant fraction was then extracted with 0.5 vol of phenol, 0.5 vol of CHCI 3 once and then I vol of CHC13 and 0.1 vol of 0.1% dextran sulphate, 0.1 vol of 5% bentonite for another two times. Finally, the supernatant was added to 0.1 vol of 3 mol of sodium acetate pH 5.2; 2.5 vols of cold ethyl alcohol absolute and stored together at - 2 0 ° C overnight. After centrifugation, the RNA found in the pellet was washed twice with 75% cold alcohol. The precipitated RNA was dried and then was resuspended in I x TE buffer (10 mmol Tris-HC1 pH 8.0 containing 1 mmol EDTA). RNA aliquots were taken for estimation of purity and content of RNA according to the ratio of spectrophotometric absorbance OD 260 nm/OD 280 nm corresponding to 1 0 D 260 = 40 #g/ml RNA.
HYBRIDIZATION ANALYSIS
Northern blot analysis The procedures of Northern blot were performed under the conditions reported by Pfeiffer and Barden (1988). Sample preparation of 5/tl RNA (10/zg) was added to a 20/tl sample denatured buffer (50% formamide, 2.2 mmol, formaldehyde, 25 mmol HEPES, 6mmol sodium acetate pH 7.0 and 1 mmol EDTA-2Na) followed by heating at 60°C for 15 min and then added to a 10/~1 sample loading buffer (20 mmol PBS pH 6.8-7.2, 50% glycerol and 0.05% Bromophenol Blue). Northern blot filters were prepared by electrophoresis of RNA sample on a denaturing 0.8% agarose gel containing 20 mmol PBS, pH 6.8-7.2, 2.2 mmol formaldehyde, 2 V cm overnight at room temperature with subsequent transfer of the separated RNA to a 0.45 # m nitrocellulose filter by passive diffusion. Filters were then air-dried for 5 min and the RNA fixed by baking at 80°C for 2 hr. These filters were then used for the hybridization test as shown below. Before transfer the control sample gel was cut down and stained with Ethidium Bromide dye. The electrophoretical migration distances (Rf) of 28S and 18S RNA were then recorded.
Dot blot analysis Three dot blots were performed for each sample. Serial dilutions of RNA 10 x SSC (8 × SSC = 155mmol NaC1, 15 mmol sodium citrate pH 7.2) were spotted on nitrocellulose filter. The RNA contents were 25, 12.5 and 6.25/~g per dot, respectively. The dot blot filter was dried in the same manner as the Northern blot filter.
Preparation of the r-prepro-ANP-cDNA probe Plasmid pNI-11 contains the r-prepro-ANP--cDNA (rANP-cDNA) sequence with approx. 650 bp which was subcloned into the PstI site of plasmid pGEM-2 (Hong and Barden, 1989). Plasmids pNI-11 were digested by PstI to recover the r-prepro-ANP-cDNA in regular methods. The ~-32P* labelled r-ANP-cDNA probe was prepared according to the programme below: 0.1 gig r-ANP-cDNA; 3/zl dGTP, dCTP, dTTP mixtures (prepared by making a 1 + 1 + 1 mixture of solution of 0.4mmol/ldGTP, 0.4 mmol/l dCTP, 0.4 mmol/d dTTP); 2/~1 of 10 × buffer; 2/zl superior to 10/tCi of (~-32P*) dATP and 3000/~Ci/mmol aqueous solution; make up to 28/al with sterilized redistilled water; 2 #1 of enzyme mixtures (DNA polymeraseI and DNAaseI in 50% glycerol). The incubation time is 35 min at 15°C. The reaction was stopped by adding 2 #1 of EDTA 0.2 mmol/1 pH 8.0. The non-incorporated deoxyribonucleoside triphosphates were removed by ethanol precipitation: add 1/10 vol of 3 mol sodium acetate pH 5.2 and 2 vol of ethyl alcohol for 2 hr at -50°C. The pellets were dissolved in an adequate amount of 1 × TE buffer.
Prehybridization, hybridization and washing The procedures used were similar under the experimental conditions reported by Pfeiffer and Barden (1988). Nitrocellulose filters were prehybridized for 4 hr at 42°C in 50% formamide, 5 × SSC, 8 × Denhardt's (50 × Denhardt's = 50% Ficoll, 5% polyvinyl pyrrolidone, 5% BSA), 50 mmol sodium phosphate pH6.5, 250/~g/ml denatured salmon sperm DNA (ss-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 × 10cpm of ~-32P*-labelled ANP-cDNA per 10ml buffer. After hybridization, filters were washed three times for 20 min at room temperature in 1 × SSC-0.1% SDS, then washed at 65°C for 1 hr and twice in 0.1 × SSC-0.1% SDS.
Autoradiography and scan of hybridization signals Finally, filters were air-dried and exposed to Kodak X-ray film with two intensifying screens at - 6 0 ° C for 7-10 days.
37
Effects of ginsenoside on ANP gene expression The optical densities of the dot autoradiography were analysed on an LKB 2202 Ultrascan Laser Densitometer. The statistical significance was analysed as reported by Iwao et al. (1988) and Mercadier et al. (1989). RESULTS AND DISCUSSIONS
The quality control analysis o f an RNA sample The ratio (OD 260nm/OD 280nm) of R N A samples extracted from tissues by the cold phenol method was more than 1.8. All the R N A samples were furthermore examined by electrophoresis on denatured formaldehyde agarose. After Ethidium Bromide staining (see Northern blot analysis), 28S and 18SRNA appeared clearly under u.v. rays (Fig. 1). These results revealed that R N A samples are not degraded. This control of non-degradated RNA is necessary for further investigation. The characteristics o f r-prepro-ANP-mRNA The resulting Northern blot assay showed that the hybridization signal of r-prepro-ANP-mRNA appeared at the front of 18SRNA (Fig. 2). It was calculated to be about 1000 b in length. This result is in agreement with those reported by Takayanagi et al. (1985) and Nakayama et al. (1984). The decline o f A N P gene expression o f elderly rats Dot blot autoradiography and densities of dot hybridization signals are shown in Fig. 3 and in Table 1. Dot hybridization analysis revealed that the relative amounts of r - A N P - m R N A in older males (14-18 months) were apparently less than those of
1
2
3
Fig. 1. (I) DNA markers: 2 DNA digested by Hind III; (2) and (3) Rat atrial RNA. A: 28S RNA; B: 18S RNA; C: 5-5.8S RNA.
m
2
3
Fig. 2. Northern blot assay: 1 = 28S RNA 3.0cm (Rf); 2 = 18S RNA 4.2 cm (Rf); 3 = ANP-RNA 4.8 cm (Rf). young rats (2-3 months), as were the female rat groups. The level of A N P - m R N A of older male rats decreased to about 14% of that in the young control group whose A N P - m R N A level was considered to be 100%. The A N P - m R N A content of older females lowered to around 60% that of the young control group (Fig. 4). Effects o f ginsenoside on A N P gene expression Dot blot hybridization signals showed that G-PSL and G-PR increased the A N P - m R N A content level of older male rats to 31 and 40% that of the young rat groups respectively, as shown in Table 1 and Fig. 4. Compared with enhancement of ginsenoside on A N P - m R N A content of older rats, ginsenoside significantly reduced the A N P - m R N A level of young male rats. The A N P - m R N A contents of G-PSL and G-PR young male rat groups were 53 and 44% that of the control young rats group, whereas neither G-PSL nor G-PR changed the A N P - m R N A contents of female rates remarkably in this experiment. The present experiment established the application of R N A Northern blot and dot blot analyses to the studies of ANP gene expression and regulation by measuring the relative concentration of A N P - m R N A . In this experiment, a distinct lower level of A N P - m R N A of older female and male (14-18 months) was observed if compared with those of young male and female (2-3 months) rats, respectively; but we wonder why the decreases of A N P - m R N A level of older male rats were much more than that of older female rats. It was worth investigating in detail. Ohashi et al. (1987) reported that the plasma h-ANP concentrations in healthy elderly men were higher than those in young men. In our experiment we originally anticipated that the level of A N P - m R N A content of older rats would be more than those of young rats. In fact, the results of
38
MINO HONG et aL
M A
B
C 1
2
3
4
5
6
F
A B
C 1 A :
25 m i c r o g r a m s .
2 B :
3
4
5
12.5 micrograms
6 & C = 6.25 micrograms.
Fig. 3. The d o t h y b r i d i z a t i o n o f r a t - a t r i a R N A : M = m a l e rats, F = female rats. 1, 2 a n d 3 g r o u p s were 2-3 m o n t h s age rats; 4, 5 a n d 6 g r o u p s were 14--18 m o n t h s age rats; g r o u p s 1 a n d 4: = c o n t r o l groups; g r o u p s 2 a n d 5: = G - P S L injected g r o u p s (50 m g / k g b o d y wt, once a d a y i.p. for 7 days); g r o u p s 3 a n d 6: = G - P R injected g r o u p s (50 m g / k g b o d y wt, once a d a y i.p. for 7 days).
our experiment were contrary to expectation, but in accord with those recently reported by Mercadier et al. (1989). These authors demonstrated that the A N P - m R N A content in atria of a 52-year-old patient free of known cardiovascular diseases was nearly 20% that of a 29 week foetus. The above results revealed in different experiments suggest that the reduction of A N P gene expression of older organisms is probably a widespread or general pathophysiological change, which happened, perhaps, before other functions including the mechanical function of the heart. Ginseng is a classical Chinese herb. It has been well demonstrated that the ginsenoside is one of the most efficacious ingredients in ginseng, which improves the function of the heart and the regulation action on blood pressure (Wang, 1985, in Chinese). In
this experiment, it was very interesting that either G-PSL or G-PR raised the A N P - m R N A content level of older male rats. The positive effects were very significant, considering the lower level of older control rats paralleled with the A N P - m R N A increase. The plasma A N P concentrations of male older rats administrated by G-PSL and G-PR also rose (not shown here). These results confirm the anti-ageing action by application of ginseng in medicine. Furthermore, no significant difference was exhibited in the regulation of A N P - m R N A between G-PSL and G-PR. In contrast to the enhancement of A N P - m R N A , both G-PSL and G-PR decreased the A N P - m R N A level of male young rats. To our surprise, no apparent effects of ginsenoside on A N P gene expression of female rats at 2-3 or 14-18 months were observed.
Table 1. The densities of dot hybridization Age (months): Group: Male rats Female rats
1
2-3 2
3
4
5
14-18 6
35.24_+0.14 20.88 _+ 1.84
18.77 -+0.48 24.48 _+0.67
15.42_+0.53 18.10 _+ 1.78
5.25_+0.36 12.45 _+0.80*
ll.01 + 0 . 6 7 " ( - 1 ) I 1.56 _ 0.91
14.31 + 0 . 8 4 * ( - 0 11.92 _+ 2.37
Note: Data represent X + SD with *P < 5% compared with group 1"-1 P < 5°/* compared with group 4; 2 and 5 were G-PSL groups: 50 mg/kg body wt once a day i.p. for 7 days; 3 and 6 were G-PR groups: 50 mg/kg body wt once a day i.p. for 7 days.
~oo-~]l~
Effects of ginsenoside on ANP gene expression
120 -% r'lxqx°'ANP'mRNA
80"
@
60"
r.~ma
40"
20" 0
1
2
3
4
5
6
Animals
Fig. 4. The relative contents of r-prepro-ANP-mRNA of different age rats and the effects of ginsenoside on preproANP-mRNA. 1:]: female rats; [l: male rats. I, 2 and 3 were 2-3 months age rats; 4, 5 and 6 were 14-18 months age rats. No. 1 and No. 4 were control groups; No. 2 and No. 5 were G-PSL groups (50 mg/kg body wt, once a day i.p. for 7 days). No. 3 and No. 6 were G-PR groups (50 mg/kg body wt, once a day i.p. for 7 days). In conclusion, in rats the expression of the A N P gene is developmentally regulated. The A N P - m R N A levels of older male and female rats were notably less than that of young rats. G - P S L and G - P R promoted the A N P gene expression of older male rats, but decreased it in male young rats. Neither G-PSL nor G - P R showed significant influence on A N P - m R N A contents of female rats. Acknowledgements--The authors would like to thank Prof. N. Barden for plasmid pNI-11. This project was supported by the National Natural Science Foundation of China. Furthermore Dr Kai-Ki Han was helped by an exchange programme between the French Medial Research Council (INSERM) and Academia Sinica for the tenure of five 3 month trips to China (1984, 1986, 1987, 1990 and 1991: Grant Nos CS-RN 739, HN-CP 1045, CS-RN 842 and 432 and CS-RN No. I). REFERENCES
Block K. D., Scidman J. G., Naftilan J. D., Fallon J. T. and Scidman C. E. (1986) Neonatal atria and ventricles secrete ANP via tissue-specific secretory pathway. Cell 47, 695-702. Espiner E. A. and Richards A. M. (1989) ANP--an important factor in sodium and blood pressure regulation. The Lancet 1, 707-710. Griffin G. D., Sellin H. G. and Novelli G. D. (1987) Optimization of phenol extraction procedures for preparation of RNA from mammalian lymphoid organs. Analyt. Biochem. 87, 506-520.
39
Hennth G. L. (1988) Physiology and pathophysiology of atrial peptide. Am. J. Physiol. 254 (Endocrinology Metabolism 17), El-15. Hong M. and Barden N. (1988) Construction of transcription system plasmid pNI-11 and pNI-12 "in vitro". J. Norman Bethune Univ. med. Sci. 15, 433-435. Iwao H., Fukui K., Kim S., Nakayama K., Ohkubo H., Nankanishi S. and Abe Y. (1988) Sodium balance effects on rein, antiotensinogen and ANP mRNA levels. Am. J. Physiol. 255 (Endocrinology Metabolism 18) E129-136. Lewicki J. A., Greenberg B., Yamanaka M., Vlasuk G., Brewer M., Gardner D., Baxter J., Johnson L. K. and Fiddes J. A. (1986) Cloning sequence analysis and processing of rat and human ANP precursors. Fed. Proc. 45, 2086-2096. Mercadier L J., Zongazo M. A., Wisnewsky C., ButlerBrown G., Gros D., Carayon A. and Schwartz K. (1989) ANP-mRNA and peptide in human heart during ontogenie development. Biochem. biophys. Res. Commun. 159, 777-782. Nakayama K., Ohkubo H., Hirose T., Inayama S. and Nakamishi S. (1984) mRNA sequence for human cardiodilatin ANP precursor and regulation of precursor mRNA in rat atria. Nature 310, 699-701. Ohashi M., Fujio N., Nawata H., Kato K. I., Ibayashi H., Kangawa K. and Matsuo H. (1987) High plasma concentration of human ANP in aged men. J. clin. Endocr. Metab. 64, 81-85. Oikawa S., Imai M., lnuzuka C., Tawaragl Y., Nakazato H. and Matsuo H. (1985) Structure of dog and rabbit precursors of ANP deduced from nucleotide sequence of cloned cDNA. Biochem. biophys. Res. Commun. 132, 892-899. Pfeiffer A. and Barden N. (1988) Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy. Molec. cell. Endocr. 55, 115-120. Scidman 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 prepro-ANP as defined by cDNA clone. Sciences 225, 324-326. Takayanagl R., Tanaka I., Maki M. and Inagami T. (1985) Effects of changes in water-sodium balance on levels of ANP-mRNA and peptide in rats. Life Sci. 36, 1843-1848. Wang B. X. (1985) The research of ginseng. First, pp. 116-118. Science and Technology Publishing House, Tian-Jin (in Chinese). Wei Y., Rodi C., Day M., Wiegand R., Needleman L., Cole B. and Needleman P. (1987) Developmental changes in rat atriopeptin hormonal system. J. clin. Invest. 79, 1325-1329. Wu J. P., Deschepper C. F. and Gardner D. G. (1988) Perinatal expression of the ANP gene in rat cardiac tissue. Am. J. Physiol. 255, E388-396. Yang-Feng T. L., Floyd-Smith G., Nemer M., Drouin J. and Francke U. (1985) The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4. Am. J. human Genet. 37, 1117-1128.
0305-0491/92$5.00+ 0.00 © 1992PergamonPresspig
THE DECLINE OF ATRIAL NATRIURETIC PEPTIDE (ANP) GENE EXPRESSION IN OLDER RATS A N D THE EFFECTS OF GINSENOSIDE ON ANP GENE EXPRESSION MING HONG,* YAN JIN,* YIN-QIAO MAI,* ARNOLD BOERSMA,?KIA-KI HAN,t~ MARIE-CHRISTINEVANTYGI-IEM§and JEAN LEWBVRE§ *Department of Biochemistry and Molecular Biology Laboratory, Norman Bethune University of Medical Sciences,Chang-Chun, Jilin Province, 130.021,People's Republic of China; ~'Unit6INSERM No. 16, Place de Verdun, 59045, Lille C6dex, France (Tel: 0033-20-52. 94.84; (Fax: 0033-20-52. 70.83); and §Departement d'Endocrinologie et M6tabolismes, USN-A, Rue du Professenr Laguesse, 59.037, Lille C6dex, France
(Received 5 July 1991) Abstract--1. The levels of atrial natriuretic peptide(ANP) gone expression in rat atria at 2-3 and 14-18 months of age and the effects of ginsenosideson r-ANP gone expression by determining the concentration of ANP-mRNA were investigated. The male and femalerats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with a-32*P-labelled r-prepro-ANP-cDNA probe. 2. The ANP-mRNA contents of 14-18 month rats were remarkably less than that of 2-3 month rats. The levels of male and female rats' atria at 14-18 months were about 15 and 60%, the content of male and female rats at 2-3 months respectively. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14-18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2-3 months. 4. These results revealed that the ANP gone expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.
INTRODUCTION Over the past decade, an important hormone has been discovered and studied in detail atrial natriuretic peptide (ANP) or named atrial natriuretic hormone. ANP is a polypeptide hormone produced by atrial heart muscle. It is a potent diuretic (natriuretic) and hypotensive agent (Espiner and Richards, 1989; Hennth, 1988). ANP is a single copy gene, which is located on the distal short arm of a human chromosome (Yang-Feng et al., 1985). The rat A N P - m R N A is nearly 800-1000 bases (Seidman e t a l . , 1984; Nakayama etal., 1984) and it is translated to give rise to a prepro-ANP. Cloning and sequence analysis of eDNA for ANP precursor show that ANP is synthesized in a prepro form containing 152 amino acids (rat) or 151 amino acids (human) (Oikawa et al., 1985). A disulphide-looped sequence of 17 amino acids with various C- and N-terminal extensions is necessary for activity. In rat and human atrial granules and homogenate, the main form of ANP is a 126-residue peptid¢ called cardionatrin IV or ),-ANP;
~/Author to whom correspondence should be addressed. Abbreviations used--ANP, atrial natriuretic peptide or atrial natriuretic hormone; G-PSL, ginsenosides prepared from ginseng stems and leaves; G-PR, ginsenosides, prepared from ginseng roots. 35
this is derived from prepro-ANP by processing. Circulating ANP appears to be a 28-residue peptide, called cardionatrin-I or ~-ANP. Both human ~-ANP and rat ~-ANP are constituted by 28 amino acid residues (Lewicki et al., 1986). The ontogenic development regulation of ANP gone expression has been thoroughly investigated in rats. In utero, both atria and ventricules produce ANP. In the newborn rats, the A N P - m R N A in atria was not detected or its level was very low, but after birth the ANP gone expression was soon activated. The activation has been demonstrated by the progressive accumulation of ANP-mRNA; simultaneously the ANP concentration in the adult atria increases 6-fold relative to foetal atria. In contrast, with ANP gone expression enhancement in atria, the levels of ventricular A N P - m R N A gradually decreased. The ventricular A N P - m R N A in adults was only 1% of that in neonatal rat (Bloch et al., 1986; Wei et al., 1987; Wu etal., 1988). Little was known about the state of ANP gene expression during ageing development. Ginseng is a classical Chinese herb, which has been used in medicine for more than 1000 years. It is therefore interesting to know whether ginseng can influence the endocrinological function of the heart or not. In this paper, we deal with the decline of A N P - m R N A levels during ontogenic ageing devel-
36
MING HONG et al.
o p m e n t a n d the effects o f ginsenoside o n A N P gene expression.
MATERIALS AND METHODS
A Nick translation kit was purchased from Boehringer Mannheim (Germany), bovine serum albumin (BSA), salmon sperm DNA (ss-DNA), restriction enzyme PstI, Ficoll, and polyvinyl pyrrolidone were purchased from the Sino-US Biotechnology Company in Beijing (China) and from the Sigma Chemical Co. (St Louis, MO). All the chemical reagents usedin these experiments were analytical grade. Most of the aqueous solutions were autoclaved or filtered by special microhole filters (Pharmacia, Sweden). Bentonite was heated at 200°C for 3 hr before use. Plasmic pNI-ll was a gift from Prof. Niocholas Barden. Ginsenosides prepared from ginseng stems and leaves (G-PSL) and from ginseng roots (G-PR) were provided by Prof. Xiang Zhou as a gift.
Animals Both male and female rats of different ages (in months) were used. They were nourished and maintained on a regular pellet diet and tap water. The male Wistar rats were 2-3 and 14-18 months in age with body wts of 200 + 20 and 380 __+40 g, respectively. Female Wistar rats at 2-3 and 14-18 months of 220 __+350 and 350 ___30 g were used for the experiment. The male rats were divided into six groups, which include groups 1-3 (2-3 months) and groups 4-6 (14-18 months). Each group had 8-10 rats. Group 1 and group 4 rats were abdominally (i.p.) injected with a suitable volume of distilled water as the control group. Group 2 and group 5 rats were i.p. injected with aqueous solution of 1% of G-PSL (50 mg/kg body wt once a day for 7 days serially) (G-PSL groups). Group 3 and group 6 rats were i.p. injected with aqueous solution of 1% G-PR once a day for 7 consecutive days (G-PR groups); the dose was the same as that for G-PSL groups. The contents of the female rat groups were identical to the male groups as described above.
Preparation of RNA samples and criteria of purity and quantity. Animals were killed at the selected time. Under ether anaesthesia, the eye globes of rats were taken out to collect blood. The atria were removed after decapitation as quickly as possible and stored in liquid nitrogen ( - 195°C) until RNA extraction, or homogenized immediately. The total RNA in atria was extracted with cold phenol methods as described by Griffin et al. (1987). Ten volumes of homogenization buffer (10 mmol sodium acetate pH 5.1; 0.1 mmol EDTA-2Na; 0.34% of SDS and 0.5% bentonite), 5 vols of phenol (which was redistilled and treated with homogenization buffer without bentonite, containing 0.1% 8-hydroxyquinoline before use) and 5 vols of chloroformisoamylalcohol (24:1, v/v) were used per weight of tissues. The above mixtures were homogenized in an ice bath and centrifuged at 4°C (10,000 g) 30 min. The supernatant fraction was then extracted with 0.5 vol of phenol, 0.5 vol of CHCI 3 once and then I vol of CHC13 and 0.1 vol of 0.1% dextran sulphate, 0.1 vol of 5% bentonite for another two times. Finally, the supernatant was added to 0.1 vol of 3 mol of sodium acetate pH 5.2; 2.5 vols of cold ethyl alcohol absolute and stored together at - 2 0 ° C overnight. After centrifugation, the RNA found in the pellet was washed twice with 75% cold alcohol. The precipitated RNA was dried and then was resuspended in I x TE buffer (10 mmol Tris-HC1 pH 8.0 containing 1 mmol EDTA). RNA aliquots were taken for estimation of purity and content of RNA according to the ratio of spectrophotometric absorbance OD 260 nm/OD 280 nm corresponding to 1 0 D 260 = 40 #g/ml RNA.
HYBRIDIZATION ANALYSIS
Northern blot analysis The procedures of Northern blot were performed under the conditions reported by Pfeiffer and Barden (1988). Sample preparation of 5/tl RNA (10/zg) was added to a 20/tl sample denatured buffer (50% formamide, 2.2 mmol, formaldehyde, 25 mmol HEPES, 6mmol sodium acetate pH 7.0 and 1 mmol EDTA-2Na) followed by heating at 60°C for 15 min and then added to a 10/~1 sample loading buffer (20 mmol PBS pH 6.8-7.2, 50% glycerol and 0.05% Bromophenol Blue). Northern blot filters were prepared by electrophoresis of RNA sample on a denaturing 0.8% agarose gel containing 20 mmol PBS, pH 6.8-7.2, 2.2 mmol formaldehyde, 2 V cm overnight at room temperature with subsequent transfer of the separated RNA to a 0.45 # m nitrocellulose filter by passive diffusion. Filters were then air-dried for 5 min and the RNA fixed by baking at 80°C for 2 hr. These filters were then used for the hybridization test as shown below. Before transfer the control sample gel was cut down and stained with Ethidium Bromide dye. The electrophoretical migration distances (Rf) of 28S and 18S RNA were then recorded.
Dot blot analysis Three dot blots were performed for each sample. Serial dilutions of RNA 10 x SSC (8 × SSC = 155mmol NaC1, 15 mmol sodium citrate pH 7.2) were spotted on nitrocellulose filter. The RNA contents were 25, 12.5 and 6.25/~g per dot, respectively. The dot blot filter was dried in the same manner as the Northern blot filter.
Preparation of the r-prepro-ANP-cDNA probe Plasmid pNI-11 contains the r-prepro-ANP--cDNA (rANP-cDNA) sequence with approx. 650 bp which was subcloned into the PstI site of plasmid pGEM-2 (Hong and Barden, 1989). Plasmids pNI-11 were digested by PstI to recover the r-prepro-ANP-cDNA in regular methods. The ~-32P* labelled r-ANP-cDNA probe was prepared according to the programme below: 0.1 gig r-ANP-cDNA; 3/zl dGTP, dCTP, dTTP mixtures (prepared by making a 1 + 1 + 1 mixture of solution of 0.4mmol/ldGTP, 0.4 mmol/l dCTP, 0.4 mmol/d dTTP); 2/~1 of 10 × buffer; 2/zl superior to 10/tCi of (~-32P*) dATP and 3000/~Ci/mmol aqueous solution; make up to 28/al with sterilized redistilled water; 2 #1 of enzyme mixtures (DNA polymeraseI and DNAaseI in 50% glycerol). The incubation time is 35 min at 15°C. The reaction was stopped by adding 2 #1 of EDTA 0.2 mmol/1 pH 8.0. The non-incorporated deoxyribonucleoside triphosphates were removed by ethanol precipitation: add 1/10 vol of 3 mol sodium acetate pH 5.2 and 2 vol of ethyl alcohol for 2 hr at -50°C. The pellets were dissolved in an adequate amount of 1 × TE buffer.
Prehybridization, hybridization and washing The procedures used were similar under the experimental conditions reported by Pfeiffer and Barden (1988). Nitrocellulose filters were prehybridized for 4 hr at 42°C in 50% formamide, 5 × SSC, 8 × Denhardt's (50 × Denhardt's = 50% Ficoll, 5% polyvinyl pyrrolidone, 5% BSA), 50 mmol sodium phosphate pH6.5, 250/~g/ml denatured salmon sperm DNA (ss-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 × 10cpm of ~-32P*-labelled ANP-cDNA per 10ml buffer. After hybridization, filters were washed three times for 20 min at room temperature in 1 × SSC-0.1% SDS, then washed at 65°C for 1 hr and twice in 0.1 × SSC-0.1% SDS.
Autoradiography and scan of hybridization signals Finally, filters were air-dried and exposed to Kodak X-ray film with two intensifying screens at - 6 0 ° C for 7-10 days.
37
Effects of ginsenoside on ANP gene expression The optical densities of the dot autoradiography were analysed on an LKB 2202 Ultrascan Laser Densitometer. The statistical significance was analysed as reported by Iwao et al. (1988) and Mercadier et al. (1989). RESULTS AND DISCUSSIONS
The quality control analysis o f an RNA sample The ratio (OD 260nm/OD 280nm) of R N A samples extracted from tissues by the cold phenol method was more than 1.8. All the R N A samples were furthermore examined by electrophoresis on denatured formaldehyde agarose. After Ethidium Bromide staining (see Northern blot analysis), 28S and 18SRNA appeared clearly under u.v. rays (Fig. 1). These results revealed that R N A samples are not degraded. This control of non-degradated RNA is necessary for further investigation. The characteristics o f r-prepro-ANP-mRNA The resulting Northern blot assay showed that the hybridization signal of r-prepro-ANP-mRNA appeared at the front of 18SRNA (Fig. 2). It was calculated to be about 1000 b in length. This result is in agreement with those reported by Takayanagi et al. (1985) and Nakayama et al. (1984). The decline o f A N P gene expression o f elderly rats Dot blot autoradiography and densities of dot hybridization signals are shown in Fig. 3 and in Table 1. Dot hybridization analysis revealed that the relative amounts of r - A N P - m R N A in older males (14-18 months) were apparently less than those of
1
2
3
Fig. 1. (I) DNA markers: 2 DNA digested by Hind III; (2) and (3) Rat atrial RNA. A: 28S RNA; B: 18S RNA; C: 5-5.8S RNA.
m
2
3
Fig. 2. Northern blot assay: 1 = 28S RNA 3.0cm (Rf); 2 = 18S RNA 4.2 cm (Rf); 3 = ANP-RNA 4.8 cm (Rf). young rats (2-3 months), as were the female rat groups. The level of A N P - m R N A of older male rats decreased to about 14% of that in the young control group whose A N P - m R N A level was considered to be 100%. The A N P - m R N A content of older females lowered to around 60% that of the young control group (Fig. 4). Effects o f ginsenoside on A N P gene expression Dot blot hybridization signals showed that G-PSL and G-PR increased the A N P - m R N A content level of older male rats to 31 and 40% that of the young rat groups respectively, as shown in Table 1 and Fig. 4. Compared with enhancement of ginsenoside on A N P - m R N A content of older rats, ginsenoside significantly reduced the A N P - m R N A level of young male rats. The A N P - m R N A contents of G-PSL and G-PR young male rat groups were 53 and 44% that of the control young rats group, whereas neither G-PSL nor G-PR changed the A N P - m R N A contents of female rates remarkably in this experiment. The present experiment established the application of R N A Northern blot and dot blot analyses to the studies of ANP gene expression and regulation by measuring the relative concentration of A N P - m R N A . In this experiment, a distinct lower level of A N P - m R N A of older female and male (14-18 months) was observed if compared with those of young male and female (2-3 months) rats, respectively; but we wonder why the decreases of A N P - m R N A level of older male rats were much more than that of older female rats. It was worth investigating in detail. Ohashi et al. (1987) reported that the plasma h-ANP concentrations in healthy elderly men were higher than those in young men. In our experiment we originally anticipated that the level of A N P - m R N A content of older rats would be more than those of young rats. In fact, the results of
38
MINO HONG et aL
M A
B
C 1
2
3
4
5
6
F
A B
C 1 A :
25 m i c r o g r a m s .
2 B :
3
4
5
12.5 micrograms
6 & C = 6.25 micrograms.
Fig. 3. The d o t h y b r i d i z a t i o n o f r a t - a t r i a R N A : M = m a l e rats, F = female rats. 1, 2 a n d 3 g r o u p s were 2-3 m o n t h s age rats; 4, 5 a n d 6 g r o u p s were 14--18 m o n t h s age rats; g r o u p s 1 a n d 4: = c o n t r o l groups; g r o u p s 2 a n d 5: = G - P S L injected g r o u p s (50 m g / k g b o d y wt, once a d a y i.p. for 7 days); g r o u p s 3 a n d 6: = G - P R injected g r o u p s (50 m g / k g b o d y wt, once a d a y i.p. for 7 days).
our experiment were contrary to expectation, but in accord with those recently reported by Mercadier et al. (1989). These authors demonstrated that the A N P - m R N A content in atria of a 52-year-old patient free of known cardiovascular diseases was nearly 20% that of a 29 week foetus. The above results revealed in different experiments suggest that the reduction of A N P gene expression of older organisms is probably a widespread or general pathophysiological change, which happened, perhaps, before other functions including the mechanical function of the heart. Ginseng is a classical Chinese herb. It has been well demonstrated that the ginsenoside is one of the most efficacious ingredients in ginseng, which improves the function of the heart and the regulation action on blood pressure (Wang, 1985, in Chinese). In
this experiment, it was very interesting that either G-PSL or G-PR raised the A N P - m R N A content level of older male rats. The positive effects were very significant, considering the lower level of older control rats paralleled with the A N P - m R N A increase. The plasma A N P concentrations of male older rats administrated by G-PSL and G-PR also rose (not shown here). These results confirm the anti-ageing action by application of ginseng in medicine. Furthermore, no significant difference was exhibited in the regulation of A N P - m R N A between G-PSL and G-PR. In contrast to the enhancement of A N P - m R N A , both G-PSL and G-PR decreased the A N P - m R N A level of male young rats. To our surprise, no apparent effects of ginsenoside on A N P gene expression of female rats at 2-3 or 14-18 months were observed.
Table 1. The densities of dot hybridization Age (months): Group: Male rats Female rats
1
2-3 2
3
4
5
14-18 6
35.24_+0.14 20.88 _+ 1.84
18.77 -+0.48 24.48 _+0.67
15.42_+0.53 18.10 _+ 1.78
5.25_+0.36 12.45 _+0.80*
ll.01 + 0 . 6 7 " ( - 1 ) I 1.56 _ 0.91
14.31 + 0 . 8 4 * ( - 0 11.92 _+ 2.37
Note: Data represent X + SD with *P < 5% compared with group 1"-1 P < 5°/* compared with group 4; 2 and 5 were G-PSL groups: 50 mg/kg body wt once a day i.p. for 7 days; 3 and 6 were G-PR groups: 50 mg/kg body wt once a day i.p. for 7 days.
~oo-~]l~
Effects of ginsenoside on ANP gene expression
120 -% r'lxqx°'ANP'mRNA
80"
@
60"
r.~ma
40"
20" 0
1
2
3
4
5
6
Animals
Fig. 4. The relative contents of r-prepro-ANP-mRNA of different age rats and the effects of ginsenoside on preproANP-mRNA. 1:]: female rats; [l: male rats. I, 2 and 3 were 2-3 months age rats; 4, 5 and 6 were 14-18 months age rats. No. 1 and No. 4 were control groups; No. 2 and No. 5 were G-PSL groups (50 mg/kg body wt, once a day i.p. for 7 days). No. 3 and No. 6 were G-PR groups (50 mg/kg body wt, once a day i.p. for 7 days). In conclusion, in rats the expression of the A N P gene is developmentally regulated. The A N P - m R N A levels of older male and female rats were notably less than that of young rats. G - P S L and G - P R promoted the A N P gene expression of older male rats, but decreased it in male young rats. Neither G-PSL nor G - P R showed significant influence on A N P - m R N A contents of female rats. Acknowledgements--The authors would like to thank Prof. N. Barden for plasmid pNI-11. This project was supported by the National Natural Science Foundation of China. Furthermore Dr Kai-Ki Han was helped by an exchange programme between the French Medial Research Council (INSERM) and Academia Sinica for the tenure of five 3 month trips to China (1984, 1986, 1987, 1990 and 1991: Grant Nos CS-RN 739, HN-CP 1045, CS-RN 842 and 432 and CS-RN No. I). REFERENCES
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Hennth G. L. (1988) Physiology and pathophysiology of atrial peptide. Am. J. Physiol. 254 (Endocrinology Metabolism 17), El-15. Hong M. and Barden N. (1988) Construction of transcription system plasmid pNI-11 and pNI-12 "in vitro". J. Norman Bethune Univ. med. Sci. 15, 433-435. Iwao H., Fukui K., Kim S., Nakayama K., Ohkubo H., Nankanishi S. and Abe Y. (1988) Sodium balance effects on rein, antiotensinogen and ANP mRNA levels. Am. J. Physiol. 255 (Endocrinology Metabolism 18) E129-136. Lewicki J. A., Greenberg B., Yamanaka M., Vlasuk G., Brewer M., Gardner D., Baxter J., Johnson L. K. and Fiddes J. A. (1986) Cloning sequence analysis and processing of rat and human ANP precursors. Fed. Proc. 45, 2086-2096. Mercadier L J., Zongazo M. A., Wisnewsky C., ButlerBrown G., Gros D., Carayon A. and Schwartz K. (1989) ANP-mRNA and peptide in human heart during ontogenie development. Biochem. biophys. Res. Commun. 159, 777-782. Nakayama K., Ohkubo H., Hirose T., Inayama S. and Nakamishi S. (1984) mRNA sequence for human cardiodilatin ANP precursor and regulation of precursor mRNA in rat atria. Nature 310, 699-701. Ohashi M., Fujio N., Nawata H., Kato K. I., Ibayashi H., Kangawa K. and Matsuo H. (1987) High plasma concentration of human ANP in aged men. J. clin. Endocr. Metab. 64, 81-85. Oikawa S., Imai M., lnuzuka C., Tawaragl Y., Nakazato H. and Matsuo H. (1985) Structure of dog and rabbit precursors of ANP deduced from nucleotide sequence of cloned cDNA. Biochem. biophys. Res. Commun. 132, 892-899. Pfeiffer A. and Barden N. (1988) Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy. Molec. cell. Endocr. 55, 115-120. Scidman 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 prepro-ANP as defined by cDNA clone. Sciences 225, 324-326. Takayanagl R., Tanaka I., Maki M. and Inagami T. (1985) Effects of changes in water-sodium balance on levels of ANP-mRNA and peptide in rats. Life Sci. 36, 1843-1848. Wang B. X. (1985) The research of ginseng. First, pp. 116-118. Science and Technology Publishing House, Tian-Jin (in Chinese). Wei Y., Rodi C., Day M., Wiegand R., Needleman L., Cole B. and Needleman P. (1987) Developmental changes in rat atriopeptin hormonal system. J. clin. Invest. 79, 1325-1329. Wu J. P., Deschepper C. F. and Gardner D. G. (1988) Perinatal expression of the ANP gene in rat cardiac tissue. Am. J. Physiol. 255, E388-396. Yang-Feng T. L., Floyd-Smith G., Nemer M., Drouin J. and Francke U. (1985) The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4. Am. J. human Genet. 37, 1117-1128.