Renal Cytochrome P4504A Activity and Salt Sensitivity in Spontaneously Hypertensive Rats
Abstract Differences in the renal metabolism of arachidonic acid by cytochrome P450 have been reported in the spontaneously hypertensive rat (SHR) and Wistar-Koyto rats, but the contribution of this system to the development of hypertension is unclear. The present study compared renal P450 activity and blood pressure in SHR and Brown-Norway rats (BN) under control conditions and in response to an elevation in sodium intake; genetic linkage analysis was performed in an F2 population (n=219) derived from these strains. Basal renal P4504A enzyme activity measured by conversion of [14C]arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) was significantly greater in the kidneys of adult SHR (n=7) than of BN (n=8) (82±7 versus 60±5 pmol/min per milligram protein). Renal 20-HETE production fell 45% in SHR and 22% in BN in which salt intake was elevated by drinking of saline instead of water for 2 weeks. Mean arterial pressure averaged 157±3 mm Hg in SHR (n=9) and 100±2 mm Hg in BN fed a normal salt diet, and it rose to 170±7 mm Hg (P<.05) in SHR and fell to 90±3 mm Hg (P<.05) in BN (n=8) after sodium intake was elevated. A polymorphic marker, D5Rjr1, that spanned a repeated element in the P4504A1 gene on chromosome 5, where all three P4504A isoforms are located, was used for genotyping of the F2 population. The P4504A genotype did not cosegregate with baseline mean arterial pressure in the F2 population; however, significant linkage was observed with the change in mean arterial pressure after sodium intake of the rats was elevated. The degree of linkage differed in male and female rats, and the highest LOD score (3.6) was observed in male F2 rats with a BN grandfather. These findings suggest that the difference in renal P450 activity in SHR and BN does not contribute to the development of hypertension in this F2 population, but it may play some role in determining the blood pressure response to an elevation in salt intake.
Renal transplantation studies have indicated that some form of renal dysfunction plays an essential role in the development of hypertension in the SHR1 2 ; however, the genetic factors contributing to the resetting of renal function and development of hypertension are poorly understood.3 The pressure-natriuretic response is blunted before the development of hypertension in very young (3-week-old) SHR.4 5 This is associated with a reduction in renal medullary blood flow and an elevation in vascular tone in the afferent arterioles of deep nephrons.6 7 The factors contributing to the elevated renal vascular resistance in this model are unknown.
Recent observations suggest that alterations in renal AA metabolism by cytochrome P450 may play a role in the development of hypertension in the SHR.8 9 In this regard, the cytochrome P4504A2 gene is selectively overexpressed in the kidneys of young SHR before the development of hypertension.10 This overexpression is associated with an elevated production of 20-HETE in the kidneys of prehypertensive SHR compared with those of WKY.11 12 Previous results indicating that 20-HETE is a potent renal vasoconstrictor that plays a role in the tubuloglomerular feedback response13 and autoregulation of renal blood flow14 suggest that elevated production of this compound in the kidney could possibly contribute to the resetting of renal function and elevation in renal vascular tone in SHR. This view is further supported by the recent observation that blockade of the renal production of 20-HETE normalizes the elevated preglomerular vascular tone in SHR kidneys15 to levels seen in normotensive rat kidneys. Reducing renal P450 activity by administration of heme oxygenase inducers has also been reported to prevent the development of hypertension in SHR.16 17 18 All of these observations suggest that abnormalities in renal AA metabolism by P4504A may play a role in the development of hypertension in the SHR; however, all of these differences in renal P4504A expression and enzyme activity may simply be strain differences that have nothing to do with the disease.
To determine whether strain differences in cytochrome P4504A metabolism of AA play a primary role in hypertension in SHR, we performed genetic linkage analysis in the present study. We identified a polymorphic marker designated D5Rjr1 spanning a tandem repeated element in intron 6 of the P4504A1 gene and used it to screen a large F2 population derived from a reciprocal cross of SHR and BN. We also confirmed that there were phenotypic differences in renal P450 activity, basal BP, and the BP response to changes in salt intake in the parental strains we used to derive the F2 population. The results indicate that the observed differences in renal expression of P4504A enzyme in SHR and BN do not contribute to the baseline BP at 18 weeks of age in an F2 population derived from these strains, but it may contribute to the differences in the BP responses to an elevation in salt intake observed between the parental strains and individuals in the F2 population.
Experiments were performed on SHR (SHR/NCrlBR) purchased from Charles River Laboratories (Wilmington, Mass). BN (BN/SsNHsd) and WKY (WKY/NHsd) were purchased from Harlan Sprague Dawley Inc (Indianapolis, Ind). The rats were housed in stainless steel cages in an animal care facility at the Medical College of Wisconsin that was approved by the American Association for Accreditation of Laboratory Animal Care. All protocols involving rats were reviewed and received prior approval by the Animal Welfare Committee at the Medical College of Wisconsin.
The rats were anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (2 mg/kg). Polyvinyl catheters were implanted in the left femoral artery with an aseptic technique.19 The catheters were tunneled subcutaneously and exteriorized at the back of the neck. The catheters were then passed through a flexible spring secured to a leather jacket that fit around the chest. The spring was attached to a swivel above the cage that allowed the rat free movement within its cage. After surgery, rats were given 40 000 U PennStrep IM (PEN-G) to prevent infection, and a 4-day recovery period was allowed before arterial pressure was measured. Arterial pressures were recorded with a Statham P23id pressure transducer with the rat resting quietly in its home cage during a 2-hour recording period. After the catheter was flushed and a 30-minute equilibration period had elapsed, heart rate, systolic BP, diastolic BP, and MAP were recorded during a 2-hour recording period. Values were then averaged over 10-minute intervals and converted to a single mean value for the entire recording period.
Phenotyping of F2 Population
F2 rats (n=219) were fed a salt diet containing 1% NaCl by weight until they were 16 weeks of age. A catheter was implanted in the femoral artery, and after a 24-hour recovery period, systolic BP, diastolic BP, MAP, and heart rate were measured during a 30-minute recording period. After these control measurements were obtained, the rats were given 0.9% NaCl to drink instead of water to raise sodium intake. After 14 days, a second catheter was implanted in the right femoral artery (if necessary) and systolic BP, diastolic BP, MAP, and heart rate were again measured during a 30-minute recording period. Previous studies20 indicated that rats achieved salt balance a few days after sodium intake was elevated, so 14 days should have provided a sufficient time to ensure that steady-state BP and sodium balance were achieved.
Phenotyping of Parental Strains
Catheters were implanted in two groups of 14-week-old adult male SHR (n=10) and BN (n=8). After a 4-day recovery period, baseline MAP was recorded for 2 hours on 3 consecutive days as described above. After this control level of arterial pressure was measured, sodium intake was elevated as was done in the F2 population by giving them 0.9% NaCl instead of water to drink. Two weeks after the rats received saline to drink, BP was again measured during a 2-hour recording period on 3 consecutive experimental days.
DNA was extracted from the liver of the F2 rats with the following protocol, modified from Liard et al.21 In brief, tissue samples were incubated overnight at 55°C in lysis buffer (100 mmol/L Tris-HCl [pH 8.0], 5 mmol/L EDTA, 0.2% sodium dodecyl sulfate, 200 mmol/L NaCl, and 200 μg/μL proteinase K). The lysate solution was then purified by phenol/chloroform/isoamyl alcohol (25:24:1). The DNA in the samples was extracted in chloroform, precipitated in isopropanol, and dissolved in TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 7.4). PCR primers were designed around a tandem repeated element in intron 6 of the P4504A1 gene obtained from a published sequence.22 The sequences of the primers used were as follows: D5Rjr1+: 5′-AAGGGAGAGAGCTGTTT-3′ and D5Rjr1−: 5′-TCTAGCCCCTGTCTGAT-3′. The forward primer was end-labeled with [32P]dATP using T4 polynucleotide kinase (New England Biolabs). PCRs were performed in a 10-μL volume with 20 ng genomic DNA using Taq DNA polymerase (Perkin-Elmer Cetus). After an initial denaturing step of 92°C for 3 minutes, samples were cycled 25 times at 92°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes. At completion of the PCR, an equal amount of formaldehyde loading buffer was added to each sample. An aliquot (3 μL) of each sample was then loaded in a 6% sequencing gel (6% Premix Acrylamide/Bis, AT Biochem). Gels were electrophoresed for 3 hours at 85 W, wrapped in plastic wrap, and exposed to x-ray film overnight. To obtain a linkage map for chromosome 5, we also genotyped the rats of the F2 population for the following flanking markers on chromosome 5: D5Mit7, D5Mit6, and D5Mit5. These PCR markers were purchased from Research Genetics.
Preparation of Renal Microsomes
Male 16-week-old SHR and BN were maintained on either a normal salt diet, 1% NaCl by weight (BN, n=8; SHR, n=7), and given water to drink or fed the same diet and given 0.9% NaCl to drink to elevate sodium intake (BN, n=8; SHR, n=10). After 2 weeks of treatment, the rats were anesthetized with pentobarbital (30 mg/kg IP), and the kidneys were rapidly removed, hemisected, and placed in an ice-cold homogenization buffer. Kidneys were also collected from very young (3-week-old) male SHR (n=6), BN (n=10), and WKY (n=6) fed a normal salt diet and given water to drink. One kidney from the adult rats and both kidneys of the young rats were homogenized in 3 mL potassium phosphate homogenization buffer (10 mmol/L, pH 7.7) containing 250 mmol/L sucrose, 1 mmol/L EDTA, and 0.1 mmol/L phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 3000g for 15 minutes to remove large pieces of tissue, and the supernatant was centrifuged at 9000g for 15 minutes, followed by 100 000g for 1 hour. The microsomal pellet was resuspended in a potassium phosphate buffer (100 mmol/L, pH 7.2) that contained 30% glycerol, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, and 0.1 mmol/L phenylmethylsulfonyl fluoride and stored at −80°C until P450 activity was determined.
Renal AA Metabolism
Renal P450 activity was measured by incubation of the microsomes (0.5 mg) with [14C]AA (0.1 μCi/mL, 20 μmol/L) in 1 mL potassium phosphate buffer (pH 7.4) containing 5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L NADPH, and an NADPH-regenerating system (10 mmol/L isocitrate acid and isocitrate dehydrogenase, 0.4 U/mL, Sigma Chemical Co). Samples were incubated at 37°C for 30 minutes after the addition of NADPH. The reactions were terminated by acidification to pH 4.0 with 0.1 mol/L formic acid, extracted twice with ethyl acetate, and dried under N2 gas, and the residue was resuspended in 500 μL of 100% ethanol. The metabolites were separated with a Hitachi 655A-1 high-performance liquid chromatographic gradient system equipped with a C18 reverse-phase column (2.1×250 mm, 5 μm; Supleco) and a 2-cm guard column (Supleco). A linear elution gradient ranging from acetonitrile/water/acetic acid (50:50:2, vol/vol/vol) to acetonitrile/acetic acid (100:0.2, vol/vol) was used. The rate of change was 1.25% per minute at a flow rate of 0.5 mL/min. Metabolites were monitored by a radioactive flow detector (Flo-one\/Beta, series A-120, Radiomatic Instruments) specially modified with lead shielding for a low background. The mean production rate for the production of each metabolite was calculated and expressed as picomoles formed per minute per milligram protein.
Renal microsomal protein (15 μg) was separated by electrophoresis on a 7.5% sodium dodecyl sulfate gel for 14 hours at 80 V. Proteins were transferred electrophoretically to a nitrocellulose membrane (Trans-Blot, Bio-Rad) at 40 V in a buffer consisting of 25 mmol/L Tris-HCl, 192 mmol/L glycine, and 20% methanol for 4 hours at 4°C. The membrane was blocked for 2 hours by immersion in a buffer (TBST-20) containing 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.8% Tween 20, and 5% nonfat dry milk. The membrane was then incubated with a 1:2000 dilution of a rabbit polyclonal antibody raised against a 20-mer synthetic peptide of the rat P4504A1 sequence that recognizes all three of the 4A isoforms.23 The membrane was washed in TBST-20 buffer and incubated for 1 hour with a 1:1000 dilution of an alkaline phosphatase–coupled goat anti-rabbit second antibody (Zymed). The membrane was then placed in a color development solution (Bio-Rad), and the relative intensities of the bands in the 50- to 52-kD range were measured with a video densitometric system (Ambis).
Values are presented as mean±SE. Significance of differences in mean values was evaluated by either a paired or an unpaired t test. A value of P<.05 was considered to be statistically significant. Quantitative trait loci (QTLs) were identified with the mapmaker program.24 An LOD score greater than 3.0 was considered significant.
Characterization of Renal AA Metabolism
Renal P4504A activity was compared from microsomes prepared from the kidneys of 3-week-old SHR, WKY, and BN by measurement of the conversion of 14C-labeled AA to 20-HETE (Fig 1⇓). The production of 20-HETE by microsomes prepared from kidneys of 3-week-old SHR was significantly greater than that seen in WKY and BN. In these very young rats, all three isoforms of P4504A protein were expressed. The elevated production of 20-HETE in the kidneys of 3-week-old SHR was also associated with increased levels of P4504A1, 4A2, and 4A3 proteins in the kidney of SHR relative to that seen in the kidney of WKY or BN (Fig 2⇓).
The renal AA metabolism in microsomes prepared from the kidneys of adult SHR and BN was also compared under basal conditions and 2 weeks after the rats were given saline to drink instead of water to elevate sodium intake (Fig 3⇓). Under basal conditions, the production of 20-HETE by the kidney of SHR was significantly greater than that seen in BN (82±7 versus 60±5 pmol/min per milligram protein). After the rats were given saline to drink to elevate sodium intake, the production of 20-HETE by renal microsomes fell by 45% in SHR, whereas it fell only 22% in BN (Fig 3⇓). In contrast to the results observed in 3-week-old rats, the main isoform expressed in kidneys of adult SHR and BN was P4504A2. The levels of P4504A2 protein were not significantly different in the kidneys of adult SHR and BN on a normal salt diet (Fig 4⇓). In addition, although the production of renal 20-HETE fell when sodium intake was elevated in both strains, the levels of the P4504A2 protein in the kidney did not decrease in either group (Fig 5⇓).
The production of other P450 metabolites of AA, such as 14,15-EET and 11,12-EET and their corresponding dihydroxyepoxyeicosatrienoic acid (DiHETE), was also compared in microsomes prepared from the kidneys of adult SHR and BN under basal conditions and 2 weeks after sodium intake was increased (Table 1⇓). Basal production of EETs was not significantly different in the kidneys of adult SHR and BN; however, the basal production of DiHETEs was significantly greater (P<.05) in the kidneys of BN than in SHR. In BN, the production of EETs was not significantly altered, but the production of DiHETEs was significantly lower in response to an elevation in salt intake (Table 1⇓).
Genetic Linkage Analysis in the F2 Population
Several PCR primers were designed to amplify around simple sequence repeated elements in various introns in the rat P4504A1 and P4504A2 genes.22 These markers were found to be highly polymorphic with the use of a panel of DNA derived from various inbred rat strains. In preliminary experiments with an F2 population derived from SHR and BN, we found that several PCR markers amplifying across repeated elements in the P4504A1 and 4A2 genes mapped to chromosome 5 within 1 to 2 cM of each other. Thus, any of these markers was equally useful for determination of linkage between P4504A genotype and BP. One of these markers, D5Rjr1, mapped to chromosome 5, 1.3 cM away from D5Mit7 and 7.7 cM away from D5Mit6. This chromosomal location was also confirmed with a somatic cell hybrid panel obtained from Dr Claude Szpirer (Université Libre de Bruxelles [Belgium]).
D5Rjr1 was used for genotyping of a large F2 population (n=219) derived from a reciprocal cross of SHR and BN. P4504A genotype failed to cosegregate with baseline MAP in the F2 generation (Table 2⇓). MAP for each genotype averaged 122±1 mm Hg in F2 rats with the SHR/SHR genotype, 125±1 mm Hg in F2 rats with the SHR/BN genotype, and 123±2 mm Hg in F2 rats with the BN/BN genotype. However, the change in MAP observed after salt intake increased was significant between the groups and averaged +6±2 mm Hg for SHR/SHR, −2±1 mm Hg for SHR/BN, and −3±2 mm Hg for BN/BN genotypes (Table 2⇓). Changes observed in BP in response to an elevation in salt intake were greater in male than in female rats (Table 2⇓). The highest LOD score (3.6) for the change in BP in response to a high salt intake was observed in a group of male rats of the F2 population with a BN grandfather (Table 2⇓). In this subgroup, BP responses of each genotype averaged +19±5 mm Hg in rats with the SHR/SHR genotype, −2±3 mm Hg in rats with the SHR/BN genotype, and −1±3 mm Hg in rats with the BN/BN genotype, suggesting a recessive mode of inheritance.
To further evaluate the possible functional significance of the linkage observed in the F2 population between P4504A genotype and changes in BP in response to an increase in salt intake, we repeated the protocol in a group of SHR and BN to determine whether the parental strains used to derive the F2 population also exhibited phenotypic differences in their BP response to an elevation in salt intake. Baseline MAP averaged 157±3 mm Hg in the SHR, and it increased significantly, to 170±7 mm Hg (P<.05), after sodium intake in the rats was elevated by giving them saline to drink instead of water for 2 weeks (Fig 6⇓). In BN, baseline MAP averaged 100±2 mm Hg, and it decreased to 90±3 mm Hg (P<.05) after sodium intake was elevated (Fig 6⇓). The difference in the BP response to a the elevation in salt intake could not be explained on the basis of differences in sodium intake between the strains. Sodium intake averaged 7.1±0.4 mEq/d in SHR and 8.2±0.2 mEq/d in the BN used in this study (Fig 7⇓).
In the present study, we performed linkage analysis in an F2 population derived from a cross of SHR and BN to determine whether cytochrome P4504A genotype contributes to hypertension in SHR. These studies were based on the results of previous studies indicating that differences in the renal AA metabolism by P450 might play some role in the development of hypertension.8 9 In this regard, the P4504A2 gene has been identified as one of two genes that is preferentially overexpressed in the kidneys of young, prehypertensive SHR.10 Previous studies have also demonstrated that 20-HETE production is elevated in the kidneys of young SHR compared with those of WKY7 11 15 and that drugs that lower renal P450 activity can prevent the development of hypertension in SHR.16 17 18
In the present study, we confirmed previous observations12 that 20-HETE production is elevated in kidneys of prehypertensive, 3-week-old SHR compared with those of WKY and extended these findings to 3-week-old BN. Using Western blot analysis, we demonstrated that these differences are associated with elevated levels of P4504A1, 4A2, and 4A3 proteins in the kidney of 3-week-old SHR relative to the levels observed in the kidneys of normotensive rats of either control strain. We also found that renal P4504A enzyme activity was higher in the kidneys of adult SHR compared with those adult BN, even though no difference in the levels of the P4504A2 protein could be detected in the kidneys of these rats at this age. Despite all of the evidence that there are strain differences in renal AA metabolism by a P4504A enzyme in the kidney of SHR and normotensive rats and the functional data suggesting that these differences might contribute to the development of hypertension, the results of the present study indicate that the P4504A genotype does not cosegregate with baseline MAP in an F2 population of rats derived from a cross of SHR and BN studied at 18 weeks of age. Although the renal production of 20-HETE was not measured in this F2 population, we did verify that there are significant differences in the renal production of 20-HETE between the parental strains. Thus, the failure to see significant linkage between BP and P4504A genotype cannot be explained on the basis of a lack of a phenotypic difference in the enzyme activity between the strains. However, it should be emphasized that failure to obtain cosegregation between P4504A and basal MAP in any single F2 population does not mean that the P4504A genotype cannot play a role in a different cross, nor does it rule out the possibility that P4504A enzymes contribute to hypertension in some indirect or permissive way in this strain.
Although P4504A failed to cosegregate with baseline MAP in the F2 population in the present cross, we did find that changes in BP after an elevation in sodium intake in the F2 population were significantly different when sorted by P4504A genotype. The greatest rise in pressure was observed in male F2 rats with a BN grandfather; in this group, the LOD score was 3.6, indicating linkage between the SHR P4504A genotype and salt sensitivity of BP in the F2 population. We also confirmed that renal 20-HETE production differed significantly between SHR and BN used to derive the F2 rats and that the the BP response to an elevation in salt intake is opposite in SHR and BN. However, additional studies characterizing P4504A activity in F2 rats derived from a cross of SHR and BN will be needed to confirm that the parental phenotypes are expressed by genotype in the F2 population. Furthermore, molecular genetic studies identifying a specific genetic difference in the P4504A gene responsible for the phenotypic difference in P4504A activity between the strains will have to be identified before one can conclude that P4504A genotype contributes to salt sensitivity of BP in SHR.
To determine whether the link between renal P450 activity and the BP response to changes in sodium intake in the F2 rats can be explained by differences in the BP response in the parental strains, we measured BP and renal P450 activities in the parental strains under basal conditions and in response to an elevated sodium intake. MAP increased significantly in SHR after sodium intake was increased, whereas MAP decreased significantly in the BN exposed to the same change in sodium intake. Overall, the increase in BP in response to an elevation in salt intake in the F2 rats inheriting the SHR/SHR P4504A genotype was about 50% of the increase in BP observed in the parental strain of SHR studied with the same protocol.
A possible explanation for the link between P4504A and the change in MAP in response to an increased sodium intake could be related to a difference in the regulation of this enzyme in the kidney between the two strains. P4504A enzyme activity in the kidney potentially influences a broad range of renal functions that can be either prohypertensive or antihypertensive. The primary AA metabolite formed by this enzyme, 20-HETE, is a potent constrictor of the preglomerular arterioles25 and mediates the autoregulatory increase in renal vascular tone in response to an increase in transmural pressure.26 It also inhibits Na+-K+-2Cl− transport in the thick ascending loop of Henle27 and Na+,K+-ATPase in the proximal tubule.28 Previous microdissection studies by Omata et al29 indicated that the enhanced renal cytochrome P4504A activity in the kidney in SHR is localized in the proximal tubule, suggesting that the elevated renal production of 20-HETE may play an adaptive role in the SHR to inhibit sodium transport in this segment of the nephron. It is possible that in the SHR the large decrease in renal P4504A activity (46% from basal levels) observed after sodium intake was elevated might blunt the inhibition in proximal tubular sodium reabsorption normally associated with the adaptation to an elevation in sodium intake. In contrast, formation of other P450 metabolites of AA such as EETs and DiHETEs in kidneys of SHR did not change significantly in response to an elevation in salt intake. Capdevila et al30 have recently reported that a marked induction of renal expoxygenases is associated with an elevation in salt intake in normotensive Sprague-Dawley rats and that this may play an important role in reestablishing salt balance. Thus, the lack of induction of epoxygenase activity in the kidneys of SHR and BN in the present study appears to indicate that induction of renal epoxygenase activity in response to an increase in sodium intake is not a general finding and it is likely that there are strain differences in the regulation of renal P450 epoxygenase by salt. The significance of this difference in regard to hypertension will have to be determined further in a genetic linkage analysis once all of the primary enzymes for the formation of EETs in these strains are fully characterized.
In summary, we performed genetic linkage analysis to determine whether the P4504A genotype is linked to the development of hypertension in a cross between SHR and BN. Despite the fact that marked differences in P4504A enzyme activity were detected in the kidneys of SHR and BN, the P4504A genotype did not cosegregate with MAP in the F2 population. However, a statistically significant linkage was observed between the P4504A genotype and the change in MAP in F2 rats after salt intake was elevated by rats drinking saline for 2 weeks. This effect was greater in male than in female rats, and the highest degree of linkage (LOD score 3.6) was observed in male F2 rats with a BN grandfather. These findings suggest that the P4504A genotype does not contribute to hypertension in an F2 population derived from a cross of BN and SHR rats studied at 18 weeks of age, but it may be a major determinant in the BP response to an elevation in salt intake observed in these two strains.
Selected Abbreviations and Acronyms
|LOD||=||logarithm of odds ratio|
|MAP||=||mean arterial pressure|
|PCR||=||polymerase chain reaction|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-36279 (R.J.R.) and grants from Bristol-Myers Squibb and the National Institute of Diabetes and Digestive and Kidney Diseases (H.J.J.). D.E.S. is the recipient of a Predoctoral Fellowship (94-pre-20) from the American Heart Association, Wisconsin Affiliate. M.R.T. is the recipient of Postdoctoral Fellowship from the American Heart Association, Florida Affiliate. J.E.K. is funded in part by grants FAPESP, FINEP4, and CNPq from Fundacro EJ Zerbini e Hemocentro of Brazil. The authors wish to thank Lisa Henderson for her excellent technical assistance.
Reprint requests to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
- Received January 15, 1996.
- Revision received February 12, 1996.
- Accepted February 22, 1996.
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