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Department of Pharmacology, University of Heidelberg (S.C., R.F., J.P.), and Deutsches Institut für Bluthochdruckforschung (T.U., S.C., J.P.), D-69120 Heidelberg, Germany; and Department of Pharmacology, University of Kiel (A.R., T.U.), 24105 Kiel, Germany
Address all correspondence and requests for reprints to: Dr. Susanne Clausmeyer, Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. E-mail: susanneclausmeyer{at}web.de
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we characterized an alternative transcript of the rat renin gene in the adrenal gland (2). In contrast to the full-length transcript coding for preprorenin, the alternative transcript lacks exon 1, containing the coding sequence of the prefragment. Instead, exon 2 is preceded by a domain of about 80 nucleotides, designated exon 1A, which originates in intron 1. The intervening sequence of intron 1, which has been excised, shows all essential sequence elements defining an as yet unidentified splice site. Thus, we concluded that this transcript, termed exon 1A-renin messenger RNA (mRNA), represents an alternatively spliced transcript, probably arising from an additional transcription start in intron 1. Our findings are supported by Lee-Kirsch et al. (3), who identified a similar transcript in the rat brain. The open reading frame of the alternative mRNA can result in a truncated prorenin, which is not targeted to the secretory pathway but remains intracellular (3). Indeed, we and others demonstrated the presence of renin within adrenal mitochondria (4, 5). Furthermore, mitochondria isolated from the adrenal gland specifically import the truncated prorenin coded by the exon 1A mRNA (2). Therefore, this form of renin might be a component of an intracellular RAS in extrarenal tissues.
These findings may change our current concepts about local renin-angiotensin systems, which have been suggested to exist in a number of tissues, such as adrenal gland, brain, and heart (6). There is evidence for a functional active RAS in the adrenal gland and the brain, where inhibitors of the RAS exert specific functions even in the absence of circulating renin (7, 8, 9). In these as well as in other tissues, expression of the renin gene has been shown (6, 10, 11). A functional RAS and local expression of the renin gene in the heart are still controversial (6). Previous analyses of the expression of the renin gene in extrarenal tissues, however, did not differentiate between the full-length transcript and the exon 1A variant. Information about the tissue-specific distribution of the two renin transcripts would be of importance to gain further insight into the function of local RASs.
We therefore decided to reinvestigate the expression of the renin gene in various rat tissues, based on RT-PCR, which allows us to discriminate between the two forms of the renin transcript. Furthermore, as our observations suggested that exon 1A-renin is the only form of renin expressed in the heart under basal conditions, we additionally analyzed the expression of the two renin transcripts in the rat heart after myocardial infarction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
RT was performed using Superscript II reverse transcriptase
(Life Technologies, Inc., Karlsruhe, Germany) with 5 µg
of each RNA and an oligo(deoxythymidine) primer containing two
degenerate nucleotide positions at its 3'-end for complementary DNA
(cDNA) synthesis.
Amplification of the cDNAs corresponding to the full-length or the
alternative transcript of the renin gene was performed by nested PCR
based on four sets of primers (Life Technologies, Inc.,
Karlsruhe, Germany); the antisense primers used for the first and
second amplifications, Ren25 [5'-GTCACTGGGTGACAGAGGAGC-3'; nucleotides
10848–10868 (13)] and Ren22 (5'-GCATGATCAACTGCAGGGAGCTG-3';
9601–9623), respectively, were identical for both transcripts (Fig. 1
). Discrimination of the cDNAs derived
from the full-length transcript and the mRNA variant was achieved by
using different sense primers; Ren6 (5'-GGAGGAGGATGCCTCTCTGGGCA-3';
40–62) and Ren24 (5'-TCAGTCTCCCGACAGACACAG-3'; 91–111) for the first
and second amplifications yielded a fragment corresponding to the
full-length transcript, whereas Ren33 (5'-CTTGAATTTCCCCAGTCAGTG-3';
3865–3885)and Ren23 (5'-GTGATGCATTGGAGGACAACTG-3'; 3883–3904)
hybridized specifically to the cDNA derived from the exon 1A-renin
transcript. Sequences complementary to the inner primers Ren24/Ren22 or
Ren23/Ren22 were separated by six introns or by five introns and about
680 nucleotides of the 3'-region of intron 1, respectively, and were
9.5 or 5.7 kb apart in the genomic DNA. Fragments derived from the
cDNAs were expected to be 855 and 849 bp in size for the inner primers
Ren24/Ren22 and Ren23/Ren22, respectively.
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Amplification was performed using Taq DNA polymerase (Promega Corp., Mannheim, Germany) starting with 2 µl of the 20-fold diluted cDNAs as a template for the first amplification over 30 cycles in a total volume of 50 µl, using the outer primer pairs. Aliquots of the first PCR reactions were diluted 50-fold, and 2 µl were then used for the following second amplification for another 30 cycles in the presence of the respective inner primer pairs. As GAP-DH is expressed to a much greater extent than renin, the primers for amplification of GAP-DH were added to each PCR after 10 cycles, so that amplification of GAP-DH ran over 20 cycles in both the first and second amplifications. Parallel samples with no reverse transcriptase served as a negative control. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. The nucleotide sequences of the fragments obtained by renin-specific primers were confirmed by direct sequence analysis.
For each tissue, samples from three animals were analyzed, and each PCR was repeated twice.
Induction of myocardial infarction and sham surgery
Myocardial infarction was induced in male SHR rats (250–300 g)
by permanent ligation of the left descending coronary artery by a
modified technique described by Johns and Olson (14). Briefly, after
induction of anesthesia with an ip injection of chloral hydrate, rats
were intubated, artificially ventilated, and connected to an
electrocardiogram recorder for continuous monitoring during surgery. A
left thoracotomy was performed by cutting the third and fourth ribs,
and a rib-spreading chest retractor was inserted. Then, the left
descending coronary artery was ligated intrathoracically with a sterile
suture (Ethibond 6–0, Ethicon, Somerville, NJ) under a
stereomicroscope. Successful ligation of the coronary artery was
verified by the occurrence of arrhythmia in the electrocardiogram and
visually by hypomotility and change of color in the ischemic area. In
rats receiving sham surgery, the ligation was placed beside the
coronary artery. Finally, the thoracic cavity was closed during
respiration hold, and analgesia was induced by sc injection of
buprenorphin-HCl (0.2 mg/kg).
Four and 5 days after coronary ligation, rats were killed, and the hearts were excised immediately. After removal of the large vessels, the atria and ventricles were separated, and tissues were immediately frozen in liquid nitrogen.
Semiquantitative RT-PCR analysis of renin transcripts after
myocardial infarction
RNA was prepared from left and right ventricles, left and right
atria, and septum of the infarcted hearts and controls as described
above. RT was performed as described, using 2 µg RNA for each
tissue.
A semiquantitative RT-PCR was performed using the respective inner
primer pairs for exon 1A-renin (Ren23/Ren22) and GAP-DH (G1/G2). First,
for both templates the number of cycles was determined, which is
required to obtain a signal visible on an ethidium bromide-stained
agarose gel and still remain in the linear range of the reaction. For
exon 1A-renin, this value was 38 cycles, whereas for GAP-DH this was
achieved at 22 cycles (Fig. 2). These
conditions were chosen for further analysis. For preprorenin,
quantification could not be performed, because there was no signal at
all.
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Tissues from four animals of each group were analyzed; each RT-PCR was repeated twice. PCR products to be compared were analyzed on the same gel. To evaluate differences between the groups, the Mann-Whitney test was performed. Only P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although the
antisense primers were identical (the outer primer being located
immediately downstream of the stop codon and the inner primer
hybridizing in exon 7), the sense primers were chosen to distinguish
both transcripts. One set of sense primers was complementary to
sequences in exon 1, and the other set hybridizes to the region of
intron 1 termed exon 1A, which is found in the alternative transcript
instead of exon 1. Therefore, using the respective pairs of nested
primers for two successive amplifications, we were able to clearly
distinguish both mRNAs. This RT-PCR, however, was not designed to
perform an exact quantification, as we were more interested in a
qualitative analysis of the tissue-specific expression of both
mRNAs.
In many of the tissues analyzed both transcripts were found (Fig. 3; summarized in Table 1), represented by a PCR product of 855
bp for the preprorenin transcript and 849 bp for the exon 1A-renin
mRNA, respectively. Both transcripts were expressed in adrenal gland,
spleen, thymus, liver, and stomach as well as in the reproductive
organs, namely uterus, ovary, and testis. There were also some tissues
of the brain, namely hypothalamus, cerebellum, medulla oblongata, and
pituitary, which contained both transcripts.
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In the aorta, thyroid gland, and submandibular gland no mRNA of the renin gene was detected.
The levels of the PCR product of GAP-DH with a size of 407 bp, which was used for an internal control, were similar in most tissues analyzed, as expected. When the GAP-DH primers were included in the parallel reactions specific for the preprorenin and the exon 1A transcript, respectively, the bands derived from GAP-DH were equivalent. In thyroid gland, submandibular gland, and intestine, the two successive amplifications of GAP-DH over 20 cycles each resulted in weak, but equal, signals.
In the negative control samples receiving no reverse transcriptase no PCR product derived from either renin or GAP-DH was seen (data not shown).
Expression of renin after myocardial infarction
The results of our RT-PCR analysis revealed the expression of exon
1A-renin, but not preprorenin in the rat heart. Earlier studies
reported an increase in renin gene expression in the heart after
myocardial infarction (15). This, therefore, raised the question of
which form of renin is induced under these conditions: exon 1A-renin,
preprorenin, or both.
Rat hearts, separated into right and left ventricles, right and left
atria, and septum, were analyzed 4 and 5 days after myocardial
infarction. On semiquantitative RT-PCR, a 4-fold increase in exon
1A-renin mRNA in the infarcted left ventricle after 4 days was observed
compared with left ventricles of sham-operated control animals, in
which a signal for exon 1A-renin was hardly visible with 38 cycles of
amplification (Fig. 4). Five days after
myocardial infarction, the amount of exon 1A-renin mRNA decreased
again. The mRNA coding for preprorenin was still not detected, even by
RT-PCR of 38 cycles (not shown) or by the nested PCR used for the
tissue-specific analysis, which comprises 2 successive PCRs with 30
cycles each (Fig. 4).
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). In contrast, in the right ventricle
(Fig. 5) and the septum (not shown) no significant changes in the
amount of exon 1A-renin transcript were found. Preprorenin mRNA was not
detectable in any of the heart tissues. No PCR product was found in the
negative control samples that did not receive reverse transcriptase
(examples shown in Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present analysis extends our recent report by demonstrating the presence of the alternative transcript in many tissues, either in addition to or instead of the known preprorenin mRNA. Both transcripts are found to be expressed, for example, in liver, spleen, hypothalamus, pituitary, testis, ovary, or adrenal gland, whereas in the kidney and intestine exclusively the full-length mRNA was detected. In the latter organs, therefore, the secretory protein appears to be the only form of renin, whereas in the former tissues an additional intracellular renin may be synthesized. Interestingly, in the heart, in both ventricles and atria, and in the lung, the exon 1A-renin mRNA proved to be the only transcript of the renin gene, whereas the transcript coding for preprorenin was absent.
The finding that in the heart exclusively the exon 1A variant is transcribed from the renin gene was rather surprising, as the current hypothesis of a local RAS in this organ is based on the assumption of the presence of a secretory renin. In the adult rat heart, renin gene expression has been investigated in many studies by RT-PCR, Northern blot, and ribonuclease protection assay (10, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23), but results were controversial. Although in several studies the presence of renin mRNA was demonstrated (15, 16, 17, 18, 19), others failed to detect any transcript of the renin gene under nonstimulated conditions (10, 11, 20, 21) or signals were nearly at the limit of detection (22, 23). Our findings clearly demonstrate the expression of the exon 1A-mRNA in the hearts of Sprague-Dawley rats under nonstimulated conditions and exclude the presence of the transcript coding for preprorenin. We suppose that in former studies demonstrating renin expression in the adult rat heart, the exon 1A variant has been detected and not the known preprorenin transcript, as the methods used were not designed to distinguish between mRNA species. Furthermore, the question arises of which transcript is expressed in the adult rat heart under stimulatory conditions, as in these studies again renin mRNA amounts have been quantified by RT-PCR using primers in exon 6 or 7 and exon 9, and this mRNA inevitably was assumed to represent the known preprorenin transcript. Stimulation of renin gene expression has been demonstrated to occur during hypertrophy (18, 19) and after myocardial infarction (15). Here, the highest concentrations of renin mRNA were measured 4 days after myocardial infarction. Days 4 and 5 after infarction were therefore chosen in our experiments to identify the renin transcript being stimulated under these conditions. Our results clearly show that the intracardiac activation of the renin gene is confined to the exon 1A-renin variant, whereas the mRNA coding for preprorenin remains undetectable. Consequently, the synthesis of preprorenin and its subsequent secretion as active renin by the heart are excluded. We did not determine which cells in the heart express the exon 1A-renin transcript; however, it has been reported that both isolated rat cardiomyocytes and fibroblasts are able to produce angiotensin I (24).
The local generation of angiotensin II in the heart has been suggested to be involved in the ventricular remodeling after myocardial infarction (25). In agreement with this hypothesis, it has been observed that myocardial infarction is followed by increased angiotensin-converting enzyme (ACE) gene expression and that both renin mRNA and ACE mRNA are localized in the border zone of the infarcted area (15, 26). Localization of renin mRNA has been shown by in situ hybridization using a probe complementary to exons 6–9, which was not specific for the preprorenin sequence (15). Our data now allow the conclusion that the renin mRNA detected in the border zone of the infarcted region corresponds to exon 1A-renin mRNA and not to the known preprorenin transcript, as exclusively the exon 1A-renin variant was detected by RT-PCR. Angiotensinogen gene expression in the heart has been demonstrated as well (15, 27). In addition, ventricular remodeling was prevented by ACE inhibition and AT1 receptor blockade (28). The specific role of intracellular renin and thus possibly intracellular generation of angiotensin in the postinfarction processes remain to be investigated. The results of the RT-PCR also show an increase in exon 1A-renin mRNA in the atria, possibly due to the volume overload, which is associated with myocardial infarction in the left ventricle and is known to activate the cardiac RAS (29, 30).
Our data on renin expression are supported by the observation that cardiac renin activity as well as concentrations of both angiotensin I and angiotensin II are increased after myocardial infarction (31, 32). However, this activation of the cardiac RAS cannot definitely be ascribed to local intracardiac synthesis of the enzyme. The analysis of cardiac renin activity is complicated by the dual origin of renin in the heart. Several findings indicate an uptake of renin into the heart from the plasma (33, 34, 35), but it has not been determined whether renin uptake is increased after myocardial infarction. Therefore, as PRA is known to increase acutely after myocardial infarction, it is conceivable, that uptake of renin from the plasma may contribute to the observed increase in intracardiac renin activity after myocardial infarction. Subcellular fractionation of normal rat hearts revealed renin activity associated with the endosomal compartment as well as the mitochondrial fraction (Peters, J., unpublished results); the latter is in agreement with our earlier observation that a protein corresponding to exon 1A-renin is transported into isolated mitochondria (2).
The exon 1A-transcript was also found to be expressed in several brain regions. In medulla oblongata, cerebellum, and hypothalamus it was detected in addition to the full-length transcript. Renin expression in these tissues has been described previously, and the presence of angiotensinogen and ACE in the brain has been proved as well (6, 10, 11, 22, 36, 37, 38). Nevertheless, evidence is still lacking that all components of a functional RAS are present within a single cell. Therefore, it has been suggested that angiotensin II in the brain is generated extracellularly. This is supported by the detection of the transcript coding for preprorenin in the present study. Although our results cannot prove the existence of a local RAS within the same cell of brain tissues, they additionally provide the possibility of an intracellular generation of angiotensins in the brain.
Our results are apparently in contrast to the findings of Lee-Kirsch et al. (3), who described the expression of the exon 1A-mRNA to be restricted to the brain, in which they did not detect the preprorenin transcript by ribonuclease protection assay. These contradictory data may be due to the higher sensitivity of the RT-PCR we performed.
Although the biochemical properties of the protein encoded by the exon 1A-mRNA are still under investigation, it is tempting to speculate that this form of renin is a component of an intracellular RAS. In those tissues expressing the preprorenin transcript as well as the exon 1A-mRNA, like adrenal gland, liver, or hypothalamus, therefore, both a secretory and an intracellular system may exist. This hypothesis is supported by the fact that in the adrenal gland renin has been detected immunocytochemically and biochemically both within cytoplasmic vesicles for storage and regulated secretion (39, 40) and within mitochondria in the zona glomerulosa (4, 41). In the adrenal, renin was detected in mitochondrial inclusion bodies of high electron density. We have also shown that these dense bodies, in contrast to the cytoplasmic granules of the kidney and adrenal gland, do not contain lysosomal enzymes, such as cathepsins (4). Interestingly, both mitochondrial renin and the number of dense bodies are increased by nephrectomy, and this is associated with an increase in aldosterone production, which partly takes place within mitochondria. Therefore, a possible role of intramitochondrial renin in the regulation of steroid biosynthesis is suggested (4, 7).
The findings presented here show that our concepts about local RASs need to be redefined, because in some systems, such as the intracardiac RAS, local expression of secretory renin is clearly excluded. In addition, our findings offer a new molecular basis for the existence of an intracellular RAS, which has been previously proposed by Mulrow (42).
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Acknowledgments |
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Footnotes |
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Received February 21, 2000.
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References |
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