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14, 1992
REGULATION OF THE EXPRESSION ANGIOTENSIN II RECEPTOR
1094-1099
OF THE RAT mRNA
Naoharu lwai and Tadashi lnagami Department
Received
of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232
December
16,
1991
Regulation of the expression levels of the rat angiotensin II receptor mRNA in the adrenal, aorta, kidney, and brain was assessed by the competitive polymerase chain reaction method. The bilateral nephrectomy or the administration of Dup753 markedly reduced the expression levels of this receptor mRNA in the adrenal and brain stem, but not in the kidney nor aorta. A continuous infusion of angiotensin II increased the expression level of this receptor mRNA in the adrenal but not in the other tissues. It is suggested that the expression level of this receptor mRNA in the adrenal is dependent on the renin angiotensin aldosterone system. 0 1992 Academic Press, Inc.
Renin angiotensin fluid homeostasis.
aldosterone The regulation
because expression
system is a prime system for the electrolytes of the angiotensin
II receptor is an important
levels of the receptor molecule will determine
regulation
polymerase
issue,
the sensitivity of the
target tissues. We have recently isolated a cDNA for the rat angiotensin receptor( 1,2 ). By applying the competitive
and body
II type I
chain reaction method, the
of the expression levels of this receptor mRNA was investigated
in the
present study. MATERIALS ..
AND
METHODS
rtrve oowcharn rmton Competitive polymerase chain reaction method to assess the expression levels of the receptor mRNA was performed as previously reported ( 2 ). The PCR products were resolved on the 5% polyacrylamide gel. The bands originated from the receptor mRNA and the deletion-mutated RNA were cut out, and the radioactivity of them were measured.
Animals
Male Spraugue-Dawley rats ( 200-2509 ) were obtained from Taconic Farm ( Germantown, NY ). Continuous angiotensin II infusion was performed by using osmotic minipumps ( model 2002, ALZET carp., Palo Alto, CA ). The pumps were filled 0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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with human angiotensin II ( Sigma, St. Louis, MO ) in a concentration of 20 ug/p.l. The rats infused with angiotensin II for 14 days became hypertensive ( more than 16OmmHg ). For the control to the bilaterally nephrectomized ,rats, uninephrectomized rats were employed. Those rats were sacrificed 24hours after the operation. Dup753 was administered ( 15 mg/kg/day ) for 3 days by dissolving in the drinking water. RNA prepTotal RNAs were extracted by guanidium isothiocyanate-cecium chloride method as previously described ( 2 ). Nothern blotting was performed , as previously reported ( 2 ). Contamination of genomic DNA was neglected by subjecting RNA samples directly to the PCR, in which no significant product was generated. Nothern blotting analysis of the 6-actin mRNA was done as previously described ( 3, 4 ). The quality of the sample RNAs were all checked by the agarose gel electrophoresis ( DMSO/glyoxal ) and ethidium bromide staining ( 3 ). Other standard molecular biological techniques were done according to Maniatis et. al. ( 3 ).
Statistics Statistical
analyses were done according to the Mann-Whittney’s
U test ( 5 ).
RESULTS the method Various amounts of the brain total RNA were mixed with the COS7 cell total RNA, in which no significant PCR product was generated by the primers used in the present study, to make the total amount of the mixture of RNAs 20 pg. And these mixtures of RNAs were combined with 200fg deletion mutated angiotensin II receptor RNA. These mixtures were reverse-transcribed using random primers as a primer, and the resultant cDNA mixtures were amplified by PCR. As shown in the Fig.1, the ratio of the radioactivity of the 479bp fragment ( originated from the angiotensin II receptor mRNA ) to that of the 191 bp fragment ( originated-from the deletion-mutated
RNA )was linealy related to the amount of the brain total RNA.
The slope of the standard curve obtained from 35 cycle amplification same as that obtained
from 40 cycle amplification
( data not shown ).
Fig.2 shows that 1.7 fold difference in the mRNA concentration by this method.
Five micrograms
( Fig. 1 ) was the
can be discriminated
of the total RNA, which contained 5pg aortic total RNA
( A ) or 3ug aortic total RNA ( B ), was combined with various amounts of the deletionmutated RNA. These two samples gave well discernable every levels of the deletion-mutated RNA.
values ( ratios of A/a ) at
The 479bp fragment generated from the adrenal total RNA was directly sequenced to confirm that the detected fragment was really originated from the angiotensin II type I receptor mRNA, cDNA for which we have cloned. 1095
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I
5.0
t
10.0
,
15-O Total RNA (pg)
Figure 1. Validity of the competitive PCR 1. 2Opg brain total RNA ( B-RNA ), 2. 2Oug BRNA+200fg deletion mutated RNA ( D-RNA ), 3. 15pg B-RNA+5pg Cos7 total RNA ( C-RNA )+2OOfg D-RNA 4. 101.19B-RNA+lOug CRNA+200fg D-RNA, 5. 7.5ug B-RNA+1 2.5ug C-RNA+ 200fg D-RNA, 6. 5.Opg B-RNA+150ug C-RNA+200fg D-RNA, 7. 2.5ug B-RNA +17.!$g C-RNA+200fg D-RNA, 8. 1.Opg B-RNA+1 9.Opg C-RNA+200fg D-RNA 9. 2Oug C-RNA+200fg D-RNA. The ratios of the radioactivity of the 479bp fragment ( A ) to that of 191bp fragment ( a ) were plotted against the amount of the brain total RNA. Each point represents a mean value of duplicate experiment.
Fiaure 2. Validity of the method Sample A containing 5pg aortic total RNA was combined with 1Opg ( lane1 ), 4pg ( lane2 ), 2pg ( lane3 ), and lpg ( lane4 ) deletion mutated RNA. Sample B containing 3ug aortic total RNA and 2ug Cos7 total RNA was combined with 1Opg ( lane5 ), 4pg ( lane6 ), 2pg ( lane7 ), and lpg ( lane8 ) deletion mutated RNA. The ratios of A/a were plotted against the reciprocal numbers of the amount of the deletion mutated RNA. 0 and 0 represent the values obtained from sample A and sample B, respectively. 1096
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Figure 3. Effects of the blockade of the RAS A. Modulation in the adrenal. Ten microgram adrenal total RNAs combined with 2pg deletion mutated RNA were reverse transcribed, and the resultant cDNA mixtures were amplified by PCR. Lane 1-3, Uninephrectomized rats ( Ux ), lane 4-6, Uninephrectomized rat administered with Dup753 ( Ux+Dup ), lane 7-9, Bilaterally nephrectomized rat ( Bx ). B. Modulation in the brain stem. Ten microgram brain stem total RNAs were combined with 200fg deletion mutated RNA. A representative of the results is shown. Lane 1, Ux, lane 2, Ux+Dup, lane 3, 6x. C. Modulation in the aorta. Ten microgram aortic total RNAs were mixed with 2pg deletion mutated RNA. Lane 1, Ux, lane 2, Ux+Dup, lane 3, Bx.
on of the receotor mRNA As shown in Fig. 3, the bilateral nephrectomy
markedly reduced the expression
level
of the receptor mRNA in the adrenal ( 15 % of the control level, pc 0.05, n=3 ) and in the brain stem ( pons+medulla
oblongata ) ( 40 % , pc 0.05, n=3). No modulation
observed in the aorta. Administration antagonist,
of Dup753, a non-peptidyl
was
type I angiotensin
II
also reduced the expression levels of the receptor mRNA in the adrenal
(50 %., PC 0.05, n=3) and in the brain stem( 60 % , p< 0.05, n=3). No modulation observed in the aorta nor in the kidney ( data not shown ). On the other hand, the prolonged
infusion of angiotensin
was
II increased the expression
level of this receptor mRNA in the adrenal ( 240 % to the control level, pc 0.05, n=4 ), but not in the brain stem, aorta, nor kidney ( Fig. 4 ). To neglect a possibility that the observed modulation
of the receptor mRNA by the
bilateral nephrectomy or by the continuous infusion of angiotensin II was due to nonspecific effect of these-stimuli, the expression levels of 6-actin mRNA were analyzed (Fig. 5 ). No significant
modulation
was observed by these stimuli.
DISCUSSION Expression levels of the angiotensin II receptor (type I ) mRNA in vivo are relatively low, which would make it difficult to assess the regulation of this receptor gene. For 1097
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. . ..,,
Ficlure
4.
Effects
of the
infusion
of angiotensin
II
Ten microgram adrenal total RNAs were combined with 4pg deletion mutated RNA. Lane l-4 , rats infused with saline only, lane 5-8, rats infused with angiotensin II.
Fiaure
5.
Expression
levels
of Gactin
mRNA
Total adrenal RNAs ( 1Oug ) were denatured ( DMSO/glyoxal ) and were electrophoresed on 1.2% agarose gel. The filter was hybridized to the 32P labelled 55 oligomer corresponding to the rat B-actin 2nd exon ( 4 ). Lane l-3, unilateral nephrectomised rats. Lane 4-6, bilaterally nephrectomised rats. Lane 7-10, control rats of saline infusion. Lane 11-14, rats of angiotensin II infusion.
of this receptor mRNA in the brain, which is one of the important target organs of angiotensin II, could not be detected by the Northern blotting method ( 2, 6 ). Therefore, we have applied the competitive polymerase chain instance,
the expression
reaction method to assess the regulation of the receptor gene. This mehtod can easily detect the receptor mRNA in the brain, and 1.7 fold difference in the mRNA concentration
can be well discriminated.
The expression
levels of the receptor mRNA were modulated
manner. The modulation
in a tissue
specific
was most markedly observed in the adrenal gland.
Either the bilateral nephrectomy
or the administration
of Dup753,
a type I specific
angiotensin II receptor antagonist, reduced the effective angiotensin II. Because these two diffrent kinds of blockade of the renin angiotensin system both reduced the expression levels of the receptor mRNA in the adrenal and in the brain stem, it is speculated that the expression levels of the receptor mRNA in these tissues may be dependent on the intrinsic activity of the renin angiotensin aldosterone system ( RAS ). As we have previously reported ( 2 ), the expression level of the receptor mRNA in the adrenal was positive& modulated by the prolonged low salt diet feeding, which is expected to activate RAS. Moreover, the continuous infusion of angiotensin II increased the expression level in the adrenal. All these experimental results strongly 1098
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suggest that the expression level of the type I angiotensin adrenal is dependent
on RAS. This positive modulation
the effects of angiotensin stem by angiotensin In summary,
RESEARCH COMMUNICATIONS
II receptor mRNA in the may be interpreted
to amplify
II. The failure to increase the expression level in the brain
II infusion may be due to the low infusion rate in the present study.
the expression levels of the rat type I angiotensin
were assessed by using the competitive
polymerase
II receptor
mRNA
chain reaction method, and the
expression level of the receptor mRNA in the adrenal was shown to be dependent the activity of the RAS.
on
ACKNOWLEDGMENT We would like to thank E.I. du Pont de Nemours & Company for providing us with DUP753. REFERENCES 1. Sasaki K, Yamano Y, Bardhan S, lwai N, Murray JJ, Hasegawa M, Matsuda lnagami T. Nature, 351 230-233,199l
M, and
2. lwai N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y, and lnagami T. Biochem.Biophys.Res.Commun. 177 299-304,199l 3. Maniatis T, Fritsh EF, and Sambrook ed. 2. Cold Spring Harbor, NY, 1989
J.
Molecular Cloning: A laboratory mannual,
4. lwai N. and lnagami T Kid. Int., 37 1480-1485,
1990
5. Siegel S. Non parametric Statistics for behavioral science. NY Mcgrow Hill Book Co. 1956 6. Murphy TJ, Alexander WR, Griendling Nature, 351 233-236,199l
KK, Runge MS, and Bernstein KE.
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