© 1998 American Heart Association, Inc.
Rapid Communication |
Kidney Is the Only Source of Human Plasma Renin in 45-kb Human Renin Transgenic Mice
From the Cardiovascular Center, Weill Medical College of Cornell University (Y.Y., R.C., T.P., J.E.S., J.H.L., D.F.C.), New York, NY; Department of Physiology, University of Iowa College of Medicine (C.D.S.), Iowa City, Iowa; and Department of Molecular and Cellular Biology, Roswell Park Cancer Institute (K.W.G.), Buffalo, NY.
Correspondence to Daniel F. Catanzaro, PhD, Cardiovascular Center, Weill Medical College of Cornell University, 1300 York Ave, Room A863, New York, NY 10021. E-mail dfcatanz{at}mail.med.cornell.edu
Abstract
Abstract—Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5'-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5'-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5'- and 8-kb 3'-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.
Key Words: transgenic • renin • gene expression • plasma renin level • nephrectomy
Several transgenic (TG) animal lines have been developed that express the human renin (hREN) gene and secrete hREN into the circulation.1 2 3 Each of these lines expresses hREN and its mRNA in the juxtaglomerular cells of the kidney, but renin mRNA is also present at many extrarenal sites not commonly associated with renin gene expression. In human subjects, circulating renin is exclusively of renal origin, whereas plasma prorenin is derived from both renal and extrarenal sources.4 Normally, an elevation in blood pressure suppresses renin secretion by the kidney.5 However, hREN TG mice6 7 and rats8 made doubly TG for the human angiotensinogen gene develop high blood pressure associated with normal to high plasma renin levels, suggesting that these TG models cannot regulate their renin secretion appropriately. Taken together, these observations suggest that circulating renin might arise from extrarenal sites where its secretion may not be regulated correctly.
In the mouse Ren-1C gene, sequences between -4.1 and -2.3 kb upstream of the structural gene appear to be required for juxtaglomerular cell expression and may also be important for the cell specificity (for reviews, see References 9 and 109 10 ). Within this region, a transcriptional enhancer was identified between positions -2866 and -2625.11 Recently, we identified a homologous sequence in the hREN gene and showed that it can stimulate transcription from the hREN promoter, albeit less strongly than its mouse counterpart.12 Because the human enhancer is located about 12 kb upstream of the transcription start site of the hREN structural gene, it was absent from previously tested hREN transgenes that contained only 3-kb1 or 0.9-kb3 5'-flanking DNA. Although all previous hREN TG mice correctly expressed hREN in the renal juxtaglomerular cells, they also expressed the hREN gene at sites not normally associated with renin gene expression. This raised the possibility that either the human enhancer or another unidentified regulatory element is required for appropriate cell-specific expression of the hREN gene in mice.
To test this hypothesis, we developed several TG mouse lines using a 45-kb NotI-SalI hREN genomic fragment that contained approximately 25-kb 5'- and 8-kb 3'-flanking DNA sequences together with all the coding and intervening sequences. Ribonuclease protection assays showed that these new 45-kb hREN TG mice display a much stricter tissue-specific expression pattern than their 13-kb counterparts, indicating that they may have a more appropriate distribution of gene expression. To determine if circulating renin was of renal origin, we examined the effect of bilateral nephrectomy on human and mouse plasma renin and prorenin concentration in 45-kb hREN mice and 13-kb hREN3 mice and their non-TG littermates. These studies showed that plasma hREN disappeared in the 45-kb hREN mice 24 hours after bilateral nephrectomy. In contrast, in 13-kb hREN mice, plasma hREN increased after nephrectomy, suggesting inappropriate secretion from extrarenal sites. Together, these observations suggest that the extended 5'- and 3'-flanking sequences in 45-kb hREN mice that were missing from previous hREN TG lines suppress renin gene expression at inappropriate extrarenal sites. Among these sites, cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma. Thus, appropriate secretion of renin requires both the correct renal expression and processing of prorenin, and the suppression of prorenin expression at extrarenal sites where it might be inappropriately processed to renin and secreted into the plasma.
Materials and Methods
Animals
All procedures involving animals were approved by the
Institutional Animal Care and Use Committee of Cornell University
Medical College. Mice were kept under standard conditions and had free
access to tap water and commercial mouse chow (No. 5008, Formulab),
which contained 0.28% sodium.
Renin and Prorenin Assays
Blood samples were obtained by orbit puncture of mice lightly
anesthetized with metaphane. Blood was collected into ice-cold
microcentrifuge tubes containing EDTA and immediately
centrifuged at 4°C to isolate plasma. Plasma was stored at
-20°C.
Plasma renin concentration (PRC) was determined by the rate of
angiotensin I (Ang I) generation from
angiotensinogen at a substrate concentration close to
Km.13 Mouse and hRENs
were distinguished by virtue of the species specificity of the reaction
between renin and angiotensinogen.14 Under the
assay conditions, mouse renin failed to generate detectable levels of
Ang I from human angiotensinogen, and the
endogenous renin in mouse plasma was unable to generate Ang
I from human angiotensinogen. Briefly, 10 µL of plasma
was incubated with 10 µL of partially purified human substrate
(
6000 ng Ang I/mL) or 50 µL of pooled plasma from 24-hour
bilaterally nephrectomized rats (3000 ng Ang I/mL) at 37°C for 1
hour or 3 hours, respectively, in a total volume of 300 µL of buffer,
pH 7.5 for mouse renin15 or pH 5.6 for
hREN.13 An unincubated blank reaction was set up for each
of the plasma samples to control for endogenous Ang I
levels. The Ang I generated was measured by
radioimmunoassay.13 PRC was calculated as the
difference between the incubated and unincubated samples. Total renin
concentration (TRC) was determined after incubation with trypsin, as
described for rat15 and human13 plasma
samples, except that no treatment was applied to remove
angiotensinogen fragments. Plasma prorenin concentration
(ProRC) was calculated as the difference between TRC and PRC.
TG Mice Generation
The transgene used was a 45-kb NotI-SalI
fragment from the P1 hREN genomic clone 3969 previously
described.12 This fragment contains about 25 kb of
5'- and 8 kb of 3'-flanking DNA sequences plus all the coding and
intervening sequences. P1 plasmid DNA was prepared by alkaline lysis of
bacterial cells from an IPTG-induced culture followed by CsCl
gradient centrifugation. The purified P1 plasmid DNA
was subjected to NotI and SalI digestion and
pulse-field gel electrophoresis. The 45-kb
NotI-SalI fragment containing the hREN genomic
sequences was excised from the gel and electroeluted. The resultant DNA
was extracted with phenol/chloroform several times, dialyzed against
injection buffer (5 mmol/L Tris, 100 mmol/L NaCl, and
100 µmol/L EDTA [pH 7.4]), and microinjected into the
fertilized one-cell embryos derived from C57BL6xCBA mice at a
concentration of 5 µg/mL. TG mice were made at the Rockefeller
University Transgenic Facility (New York, NY).
Mouse Genomic DNA Analysis
Mouse genomic DNA was extracted from tail tips by
digestion with proteinase K (0.5 mg/mL) in the lysis buffer (4.0 mol/L
urea, 5 mmol/L CDTA, 0.5% Sarkosyl, 0.2 mol/L NaCl, and 0.1 mol/L
Tris [pH 8.0]) at 55°C with rocking overnight. To 450 µL of
supernatant, 1 mL 100% ethanol was added and rocked manually until the
mouse chromosomal DNA aggregated into a visible clump. The DNA was then
spooled with a sealed hematocrit tube and dissolved in 0.5 mL
0.1xTE (10 mmol/L Tris-Cl, 1 mmol/L EDTA; pH 8.0) by rocking
for about 30 minutes. The DNA concentration was estimated by visual
comparison to size markers of known concentration on a 1.0% agarose
gel. Southern blots to identify founder mice were carried out with 10
µg of DNA from each mouse digested with several different restriction
enzymes. Blots were probed with a full-length hREN cDNA16
or the hREN enhancer12 made by random primed
synthesis.
RNA Preparation and Ribonuclease Protection Assay
Mice were killed by spinal cord dislocation and the tissue
samples were removed immediately, frozen in liquid nitrogen, and stored
at -70°C. Tissues were homogenized with a polytron
homogenizer in Ultraspec RNA reagent (Biotecx
Laboratories, Inc, Houston, Tex), and total RNA was extracted according
to the manufacturer's protocol. Ten micrograms of total tissue RNA was
hybridized to single-stranded labeled antisense RNA probes generated
using the Maxiscript kit (Ambion, Inc). Ribonuclease protection assays
were carried out using the RPAII kit (Ambion, Inc). Protected fragments
were separated by electrophoresis through 5%
polyacrylamide/urea sequencing gels and visualized by
autoradiography. Quantification was carried out by
phosphorimaging.
A set of short riboprobes was designed to detect both mouse and hREN. Regions were selected with maximal homology between human and mouse renin cDNA. The sequences used were hREN cDNA residues 550 to 730 (in the coding sequence) cloned into pCRII (Invitrogen) and mouse Ren-1C cDNA residues 184 to 418 cloned into pBSKS(-) vector. A longer hREN riboprobe spanning residues 741 to 1148 was also used.17 Probes for mouse ß-actin and 18S rRNA were made with templates supplied with the RPAII kit. The specific activity of the 18S rRNA probe was reduced by adding 0.5 mmol/L unlabeled UTP and half the usual volume of 32P-UTP to the reaction mixture. All probes were added to molar excess evidenced by the lack of any further increases in signal strength with greater amounts of probes. Size markers were produced using the Century Marker Template Set (Ambion, Inc).
Immunocytochemistry
Tissues were placed in Bouin Fixative (Poly Scientific) for 12
to 24 hours at room temperature. After fixation, the specimens were
transferred to 70% ethanol for >2 hours, embedded in paraffin, cut
into 5-µm sections, and mounted on slides. The sections were dewaxed,
rehydrated, and washed before incubation with the primary antibody at
4°C overnight. Bound antibody was detected by light immunogold silver
staining using AuroProbe LM and IntenSE M kits (Amersham). The primary
antibodies used were BR1 -5,15 R15,18 and F37
2D12.19 BR1–5 and R15 are polyclonal rabbit anti-hREN
antibodies, and F37 2D12 is mouse monoclonal anti-hREN antibody. The
dilution factors used for each of the antibodies were 1:1000, 1:500,
and 1:21, respectively.
Results
Characterization of TG Mice
To determine whether the extended flanking sequences surrounding
the hREN structural gene would restrict hREN gene expression to fewer
sites, TG mice were constructed using a 45-kb
NotI-SalI fragment isolated from the P1 clone
396912 (Figure 1A
). The
45-kb NotI-SalI fragment contains about 25-kb 5'-
and 6-kb 3'-flanking DNA sequences plus all the coding and intervening
sequences.
|
Forty mice were obtained from 8 surrogate mothers, of which 6 (15%)
contained the transgene, and 3 transmitted the transgene to their
offspring. All lines were maintained heterozygous for the transgene.
Southern blot hybridization of KpnI and PstI
digests of mouse genomic DNA with probes containing the hREN cDNA or
enhancer sequences showed that each line contained intact copies of the
structural gene and the 5'-flanking DNA containing the enhancer element
(Figure 1B
). All 3 lines gave the same hybridization pattern as
the P1 3969 clone with either the cDNA or enhancer sequence probes.
Notably, the contiguous KpnI fragments that contained the
enhancer, 5'-flanking DNA, coding, and intervening sequences were
present in each TG line. By comparison to the hybridization signal
produced by the endogenous mouse renin gene in this and
other experiments, it was estimated that line No. 3 contains one copy
of the transgene and line Nos. 2 and 18 contained 4 and 6 copies,
respectively (see Figure 1B; PstI digest hybridized
with hREN cDNA).
Tissue Distribution of hREN mRNA
Tissue specificity of hREN transgene expression in the mice was
addressed at the mRNA level using a ribonuclease protection assay.
Figure 2 shows the tissue distribution of
human and mouse renin mRNAs in a mouse from the 45-kb hREN No. 2 line.
Both mouse and hREN mRNAs were detectable in the kidney, ovary,
adrenal, and eye of TG mice, and mouse, but not human, renin mRNA was
present in the submandibular gland. hREN mRNA was also detected at
high levels in the lung and at lower levels in the brain, although no
mouse renin mRNA was detected at either site. Similar results were
obtained with mice from No. 3 and No. 18 lines.
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The tissue distribution of hREN mRNA in 13-kb hREN mice (No. 10 line)
was also examined. A representative ribonuclease
protection assay is shown in Figure 3. In
addition to the kidney, adrenal ovary, and adipose tissue reported
previously,3 abundant hREN mRNA was detected in the eye,
skeletal muscle, and stomach, and lower levels were detected in brain,
small intestine, and spleen. However, unlike the earlier
report,3 no hREN mRNA was detected in testis or
submandibular gland.
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Renal renin mRNA levels were determined for each of the three 45-kb
lines and a 13-kb hREN line (No. 10) (Figure 4). Among 45-kb hREN lines, hREN mRNA
levels in the kidney were proportional to the copy number (Figure 5).
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Immunocytochemistry
The cellular localization of hREN transgene expression was
examined in the kidney and lung by immunocytochemistry using antirenin
antibodies (Figure 6, top). BR1–5, which
stains both mouse and hREN, and prorenin stained the
juxtaglomerular apparatus (JGA) and afferent
arterioles of both TG and non-TG kidneys. However, R15, which is highly
specific for hREN, stained only JGA from TG mouse kidneys. All JGA of
TG animals that stained with BR1–5 also stained with R15, suggesting
that there is no differential expression of mouse and hRENs between
nephrons. Similar results were obtained with kidneys from the other two
45-kb hREN lines in which the intensity of staining with R15 was
proportional to the plasma hREN levels (not shown).
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The immunocytochemical staining on the adult TG mouse lung with
antibody F37 2D12 showed hREN expression in epithelial cells of alveoli
and to a lesser extent in isolated venous endothelial
cells (Figure 6, bottom). No specific staining was detected in
the lung of non-TG littermate using the same antibody.
Plasma Levels of Mouse and hREN and Prorenin
Plasma levels of mouse and hREN and prorenin in the various 45-kb
hREN and 13-kb hREN TG lines and their non-TG littermates are shown in
Table 1. Human PRC (hPRC) and
ProRC differed between the TG lines and were proportional to the copy
number (Figure 5) among the 45-kb hREN lines. The proportion of
the total renin (renin+prorenin) detected as renin was about 56% in
line No. 2 and No. 3 and slightly lower (47%) in line No. 18. The
proportion of hREN in 13-kb hREN mice was much lower (6%), because the
human plasma prorenin was about 10x higher than in 45-kb hREN mice.
Mouse PRC and mouse ProRC were similar between TG lines. Mouse PRC
tended to be lower in TG mice than in their non-TG littermates, but
this difference was statistically significant only for the No. 3 line.
Plasma ProRC accounted for 4% to 10% of the total mouse renin in
45-kb hREN mice but was undetectable in 13-kb hREN mice.
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Nephrectomy Studies to Determine the Source of Circulating
hREN
To determine whether plasma hREN originates from the kidneys,
bilateral nephrectomy was carried out on mice from each of the TG lines
(Figure 7).
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After nephrectomy, mouse plasma renin decreased 50- to 70-fold to very low levels in every mouse, irrespective of the TG status or line. Human plasma renin also fell in each of the 45-kb hREN lines to levels indistinguishable from their non-TG littermates. In marked contrast, in the 13-kb hREN mice, human plasma renin levels increased almost 6-fold, despite the dramatic reduction in the mouse plasma renin levels. The increased renin activity in 13-kb hREN mice after nephrectomy was inhibited by the renin inhibitor Hui Pep-27 and by the antirenin antibody BR1–5 (not shown). These data indicate that the increased renin activity is indeed due to the release of hREN.
Both mouse and human prorenin levels were unaffected by nephrectomy and in some cases actually increased, indicating that most, if not all, of the circulating prorenin in these mice originates from extrarenal sites.
Discussion
In the present study, we generated 3 mouse lines carrying a 45-kb hREN genomic fragment. These mice express the hREN transgene in a much narrower range of tissues than previous hREN TG lines and are unique in that they secrete hREN into the circulation exclusively from the kidneys, as shown previously in normal mice and humans.20 21
hREN mRNA was detected in 45-kb mice only in the kidney, eye, ovary,
brain, adrenal, and lung. With the exception of lung (see below), this
tissue distribution of transgene expression more closely resembles the
pattern in normal human and animal tissues18 22 23 24 25 26 27 than
previous mouse and rat lines (Table 2).
Although at least some of the extrarenal sites of transgene expression
in earlier models were interpreted to represent previously
unidentified sites of hREN gene expression,28 failure to
replicate expression at these sites with a longer transgene suggests
that they are actually inappropriate sites of ectopic expression.
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Ectopic expression most likely explains the surprising increase in circulating human plasma renin after bilateral nephrectomy of 13-kb hREN mice, even as mouse renin disappeared. Ectopic expression of prorenin in cells that express a processing protease—to which prorenin would normally never be exposed—could lead to its conversion to renin and its secretion into the plasma. However, we do not know whether, under normal circumstances, extrarenal sites in 13-kb hREN mice actually contribute to the circulating renin. Previous observations of both appropriate and inappropriate responses of circulating hREN to physiological and pharmacological stimuli in 13-kb hREN mice17 suggest that not all of their circulating hREN originates from the kidney. In contrast, in 45-kb hREN mice, hREN mRNA and plasma hREN levels respond appropriately to changes in dietary salt, angiotensin-converting enzyme inhibition, ß1-adrenergic stimulation, and infusions of angiotensinogen that increase blood pressure and/or circulating levels of Ang II.29 Taken together, these observations indicate that for appropriate processing and secretion to occur, prorenin expression must be prevented at sites where cryptic processing of prorenin could lead to abnormal renin secretion into the plasma.
Plasma hREN levels in the three 45-kb hREN lines ranged from 9 to 66 ng of Ang I per mL/h and correlated with the transgene copy number, which ranged from 1 to 6. These levels are very low compared with the endogenous mouse renin concentration (>200 ng Ang I per mL/h). In the No. 3 line that contains a single copy of the hREN transgene, the hPRC was around 9 ng Ang I per mL/h, which is very similar to the hPRC in normal human plasma (2 to 15 ng Ang I per mL/h).13 This result suggests that the plasma renin levels are determined by sequences within the renin gene, ie, they are encoded in cis. This could be due to differences in transcription rate, mRNA turnover, or differences in the rate of renin synthesis, secretion, or degradation and clearance. Our recent studies suggest that the mechanism is posttranscriptional.29
Plasma hREN and renal hREN mRNA levels in 13-kb hREN mice were similar to the 45-kb hREN mice. It therefore seems unlikely that the extended sequences in the 45-kb hREN transgenes contribute significantly to expression levels. Despite the broad tissue distribution of transgene expression in earlier studies, hREN was correctly localized in the juxtaglomerular cells of the kidney,1 2 3 suggesting that the shorter transgenes contain all the sequences required for juxtaglomerular cell-specific expression. Therefore, the enhancer contained in the extended 5'-flanking sequence of the 45-kb hREN transgene is probably not required either to direct hREN expression to renal juxtaglomerular cells or to determine the level of expression. This is consistent with the relatively weak transcriptional activity of the human enhancer observed in transfection experiments12 and suggests that if the enhancer plays a role in directing hREN expression in vivo, then its effects are mediated outside the kidney. Thus, the enhancer or some other sequences in the 45-kb hREN transgene may function to restrict expression to appropriate physiological sites. Transcriptional suppression at inappropriate sites of expression has been described for the IgH enhancer.30
The plasma human prorenin levels were similar (47% to 56% of total
renin) among the three 45-kb lines but differed from 13-kb hREN and
other hREN TG lines in that the absolute level and the proportion of
total renin were much lower.2 31 The high plasma human
prorenin level in 13-kb hREN mice that persisted after nephrectomy is
consistent with the broad tissue distribution of hREN
expression in this line, suggesting that many of these extrarenal sites
secrete prorenin into the plasma. Although prorenin is normally about
90% of total human plasma renin, the proportion that we measured in
mice was lower (0% to 16%). The mouse prorenin levels we measured are
similar to values derived from data for C57BL/6J
(7%),31 although in Balb C mice,32 the
prorenin levels were 90%. In other studies of the 45-kb hREN No. 2
line backcrossed for at least 5 generations to C57BL/6, we measured
mouse prorenin levels at 50% of the total, whereas the hREN levels
were similar to those reported in the present study.
Because prorenin is a derived value, we cannot be certain of the absolute levels of prorenin in mouse plasma when it comprises <10% of the total plasma renin. Nevertheless, some important conclusions can be drawn from the relative differences between TG lines and treatments. After nephrectomy, mouse and human plasma prorenin levels in 45-kb hREN were unchanged or were slightly increased (<2-fold). This indicates that plasma prorenin may arise from extrarenal sites. However, it is also possible that prorenin secreted by kidney disappears after nephrectomy and is replaced by an increase in prorenin secreted from extrarenal sites.
hREN gene expression was detected in the lung of each of the 45-kb hREN
lines. However, the lung does not appear to process prorenin to renin
and to secrete renin into the circulation of 45-kb hREN TG mice,
although it may be a source of circulating human prorenin. hREN gene
expression was also detected in the lung of all other hREN TG lines
(Table 2) and has been reported in human fetal
lung.33 However, in each case, the cellular localization
differed: in the present study, we found hREN immunoreactivity
primarily in bronchiolar epithelial cells; in 13-kb TG mice, hREN mRNA
was localized to type II epithelial cells34 ; and in human
fetal lung, renin was localized to endothelial
cells.33 Renin has also been detected in human
pulmonary tumors35 36 37 and in a human
pulmonary carcinoma cell line (Calu-6).34 Because
lung neoplasms ectopically express many other polypeptide
hormones,38 lung tissue may more readily express a variety
of genes, and renin gene expression may be readily activated if
all the necessary regulatory mechanisms are absent or compromised. This
could result from some incompatibility between the mouse
transcriptional mechanisms and the hREN genomic sequences that normally
prevent pulmonary expression or from the lack of essential
sequences that are absent even from the 45-kb hREN transgene. This
raises the possibility that in renin-secreting tumors of extrarenal
origin, renin gene expression might be activated by loss of
either the required regulatory sequences through mutation or genetic
rearrangement or the trans-acting factors that normally
interact with these sequences to prevent expression. Alternatively,
some cryptic activation mechanism could result in transcription of the
hREN transgene in mouse lung.
In summary, our new 45-kb hREN TG lines show copy-number dependent expression in renal juxtaglomerular cells and extrarenal expression restricted to far fewer tissues than previous models. Importantly, in 45-kb hREN mice plasma, hREN is derived exclusively from the kidneys, as shown previously in humans and other species. We conclude that the additional sequences contained in the 45-kb hREN gene restrict hREN gene expression at inappropriate sites. These new TG mice should provide a better animal model for the study of the expression and regulation of the hREN gene, the regulation of renin secretion, and the role of renin in the pathophysiology of hypertension and vascular disease.
Acknowledgments
This work was supported by NIH grants DK45982 (D.F.C.), HL48459 (K.W.G.), and HL48058 (C.D.S.) and by the generous support of the Greenburg, Wallace, and Wolk Funds (D.F.C.). Daniel F. Catanzaro and Curt D. Sigmund are Established Investigators of the American Heart Association.
Received August 14, 1998; accepted October 13, 1998.
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