Nitric oxide is thought to be involved in blood pressure regulation. Nitric oxide synthase (NOS) genes are logical candidates for genetic hypertension. Of the three known forms of NOS, the “neuronal” and “inducible” Nos genes have been tested as candidate genes for causing inherited hypertension in Dahl salt-sensitive rats. In the present work, we analyzed the endothelial Nos gene, designated Nos3, directly and indirectly for cosegregation with blood pressure in six F2 populations independently generated from crosses of Dahl salt-sensitive rats with rats of various other strains. The Nos3 alleles did not cosegregate with blood pressure in these populations. Therefore, Nos3 is an improbable, if not impossible, candidate gene for causing hypertension in the Dahl salt-sensitive rat.
There are at least three different types of NOS genes. Designated Nos1, Nos2, and Nos3, they code for the “neuronal” NOS, “inducible” NOS, and “endothelial” NOS, respectively.1 Nos1 and Nos3 are single genes, whereas Nos2 is made up of a multiple gene family. In the rat, the Nos2 gene family, at a minimum, consists of genes of the vascular smooth muscle and macrophage subtypes2 and also includes a Nos2 gene with a 3-bp deletion in the coding region.3 Nos1 and Nos3 are localized to rat chromosomes 124 and 4,5 respectively. Nos2 genes are localized to rat chromosome 10.6 7
Pravenec and associates8 found interleukin-6 (IL6) alleles significantly cosegregated with mean arterial pressure in 36 recombinant inbred strains derived from crosses of SHR and BN rats. Because Nos3 is located close to IL6 (see Reference 5 and this work), Nos3 could be viewed as a potential candidate gene for a QTL for BP. In human genetic hypertension, it is uncertain what role, if any, Nos3 plays because Nos3 was not linked to essential hypertension in one human population9 and a negative linkage result from the analysis of one human population was not enough to rule out Nos3 as a BP QTL in other human populations.10
Up to now, genetic variations in Nos1 and Nos2 have been tested for cosegregation with BP in F2 populations originating from crosses of the inbred hypertensive Dahl salt-sensitive rat with various rats of other strains.7 This report documents the data from genetic analyses of Nos3 alleles for cosegregation with BP in several F2 populations.
Animals and methods of BP measurement were the same as described previously.7 Inbred Dahl salt-sensitive (SS/Jr) and inbred Dahl salt-resistant (SR/Jr) rats were bred in our own colonies and are designated as S and R, respectively. The other rat strains used in the present work were SHR and WKY obtained from Harlan Sprague Dawley, Inc (Indianapolis, Ind); Milan normotensive rats (MNS) from Veterinary Resources Branch, National Institutes of Health (Bethesda, Md); Lewis rats (LEW) from Charles River Laboratory (Wilmington, Mass); BN rats from Harlan Sprague Dawley (Indianapolis, Ind); and Albino surgery rats (AS) from the National Institute for Medical Research (Mill Hill, UK). To generate segregating F2 populations, we crossed S rats with various contrasting strains and intercrossed the F1 offspring to produce F2 rats.
F2 rats were weaned at 30 days of age and fed on a high salt (8% NaCl) diet (TD82050, Teklad) starting at 37 days of age. Systolic BP was measured by the tail-cuff microphonic manometer method with the rats warmed to 28°C either under light ether anesthesia or in the conscious, restrained state (IITC Inc). When the highest BPs of rats in the population reached more than 200 mm Hg, the BP of the entire population was measured intensively during a 7- to 10-day period. At least three consistent BP readings at a given session were acquired for each rat and averaged as that session's reading. Three such separate sessions on different days were conducted for each rat. The BP from three sessions was then averaged and taken as the final BP measurement of that rat. All F2 rats presented in the current work were males.
Table 1⇓ lists chromosome markers used in the present study. These markers were genotyped by PCR.7 The PCR conditions were the same as described previously.7 Briefly, the PCR program was run on a Thermocycler (MJ Research). The PCR cycles for primers were as follows: 95°C for 5 minutes, 30 cycles of 94°C for 40 seconds, 55°C for 40 seconds, and 72°C for 1.5 minutes, with extension for 5 minutes at 72°C.
Statistical and Linkage Analysis
Statistical analyses were carried out by one-way ANOVA with SPSS programs that compared the BP of rats among differing genotypes for each locus. Linkage analyses were done with MAPMAKER programs.13
Using a Nos3 marker composed of simple-sequence repeats (SSRs),5 we initially genotyped Nos3 in the F2 S×LEW and F2 S×SHR populations. The results showed that Nos3 alleles did not cosegregate with BP in either population (Table 2⇓). Since our previous work demonstrated that the outcome of cosegregation of a chromosome marker depends on the F2 population used,14 genotyping one or two F2 populations when the results are negative does not adequately constitute a meaningful test of the marker of interest for cosegregation with BP. For example, the locus alleles for the angiotensin-converting enzyme (Ace) locus did not cosegregate with BP in the F2 S×WKY population but did cosegregate with BP in the F2 S×MNS population.14 It would have been erroneous to rule out Ace as a marker for the region of rat chromosome 10 potentially containing a BP QTL solely because Ace alleles did not cosegregate in F2 S×WKY rats.
It turned out that the available Nos3 marker5 is not polymorphic between the S and WKY, R, MNS, AS, or BN strains (Table 1⇑). As a consequence, we could not directly analyze Nos3 in F2 populations derived from crosses of S rats with rats of these strains. However, several SSR markers are reported to be linked to Nos3.5 11 We reasoned that because markers immediately proximate to the locus of interest always show similar statistical profiles of segregation with BP as the locus of interest per se,7 10 15 16 17 the markers in such close vicinity to Nos3 (ie, within 1 to 2 cM) can validly surrogate for Nos3 in cosegregation analysis.
Table 1⇑ lists SSR markers used in our genetic analyses for Nos3. Of these markers, D4Bro1 was reported by other investigators11 to be closely linked to D4Mgh22 and IL6 on rat chromosome 4. But when we genotyped D4Bro1 in two of our F2 populations, ie, F2 S×R (n=112) and F2 S×WKY (n=159), it was actually linked to the inhibin (Inh) locus by less than 2 cM. It is well established that Inh is located on rat chromosome 9.6 12 We further noted that D4Bro1 was described as a marker for the Aep3 gene,11 which is probably the same gene, ie, the cardiac AE-3 Cl−/HCO3− exchanger (Ae3) gene. The Ae3 gene with the GenBank Accession number M87060 has been previously mapped to chromosome 9 both by analyzing rat-mouse somatic hybrids and by linkage.6 Both Ae3 and D4Bro1 were mapped to the same chromosome position in both F2 S×R and F2 S×WKY populations in the present work. Thus, the assignment of D4Bro1 to rat chromosome 411 was incorrect.
To authenticate the close linkage relationships between Nos3 and either D4Mgh22 or IL6 as reported in the literature,5 11 we evaluated these markers not only for cosegregation with BP but also for linkage among them in our crosses. Nos3 and D4Mgh22 are 1.0 cM apart in F2 S×LEW (n=151). D4Mgh22 is 1.1 cM away from IL6 in F2 S×BN (n=96). Therefore, both D4Mgh22 and IL6 are closely linked to Nos3.
As shown in Table 2⇑, Nos3 has been tested in a total of six F2 populations and thus far is not supported as a candidate gene for causing BP differences between the S rats and the rats of any of the strains tested, ie, AS, SHR, LEW, BN, MNS, and WKY.
The results presented in the current work complete the overall genetic assessment for the rat Nos loci in genetic hypertension of the S rat. Of Nos1, Nos2, and Nos3 loci, only the alleles of the Nos2 locus cosegregated with BP.7 This indicates that a BP QTL is present on rat chromosome 10 marked by Nos2 in the S rat. Although our genetic tests for Nos17 and Nos3 (present work) were by no means exhaustive, no evidence implicates either Nos1 or Nos3 as being a BP QTL in the S rat.
Gene “knockout” experiments in the mouse have been carried out for each of the three Nos genes one at a time. Results showed that the homozygous mice lacking Nos1 alone, when Nos2 and Nos3 were intact, had BP similar to that of wild-type mice.18 Thus, the involvement of Nos1 in genetic hypertension is not clear. The homozygous mice deficient only in Nos2 did not show a drastic decrease in BP in response to injection of lipopolysaccharides, as did the wild-type mice.19 This result implies that Nos2 may have a BP-lowering effect. Whether the BP of the mice lacking Nos2 would be different from that of wild-type mice without lipopolysaccharide induction remains to be determined.19 20 However, another research group21 working with an independent Nos2 knockout did not see a BP-lowering effect such as did MacMicking and associates.19 In mice missing only Nos3, their BP was significantly higher than that of wild-type mice,22 suggesting that Nos3 had a BP-lowering effect.
Nevertheless, the mouse models with these Nos gene knockouts cannot be directly applied to understanding the genetic defects causing hypertension in the S rat. The S rat possesses all three Nos genes, which are intact, expressed, and functional in, for instance, kidneys.3 23 If there could be any genetic defect in any of the Nos genes of the S rat causing hypertension, it would have to be an alteration in a Nos gene that results in variations in the function of the gene product either in quantity or quality, not because of a deficiency in the gene product.
The study of animal models of hypertension inevitably evokes the issue of how useful they are in helping to disclose multifaceted mechanisms of human genetic hypertension. With regard to Nos3, it was not supported as a valid candidate QTL for the inbred Dahl rat model (present work) but was somewhat implicated as a candidate QTL in the SHR model.8 This strain dependence of animal studies may encourage further studies of NOS3 in more human populations despite the negative result from one human population.9
Selected Abbreviations and Acronyms
|BN||=||Brown Norway (rat)|
|NOS||=||nitric oxide synthase|
|PCR||=||polymerase chain reaction|
|QTL||=||quantitative trait locus|
|S||=||Dahl salt-sensitive (rat)|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by a Grant-in-Aid from the American Heart Association; by a Grant-in-Aid from the American Heart Association, Ohio Affiliate to A.Y. Deng; and grants from the National Institutes of Health and Helen and Harold McMaster Chair to J.P. Rapp.
- Received April 19, 1996.
- Revision received May 24, 1996.
- Revision received July 25, 1996.
Nathan C, Xie Q. Regulation of biosynthesis of nitric oxide. J Biol Chem. 1994;269:13725-13728.
Deng AY. Molecular analysis of the rat inducible nitric oxide synthase genes. Hypertension. 1995;26:576. Abstract.
Deng AY, Rapp JP, Kato H, Bihoreau M. Linkage mapping of the neuronal nitric oxide synthase gene (Nos1) to rat chromosome 12. Mamm Genome. 1995;6:824.
Hübner N, Kreutz R, Rubattu S, Lee YA, Ganten D, Allen PD, Lindpaintner K. The gene encoding endothelial nitric oxide synthase, Nos3, maps to rat chromosome 4. Mamm Genome. 1995;6:758-759.
Pravenec M, Gauguier D, Schott J-J, Buard J, Kren V, Bila V, Szpirer C, Szpirer J, Wang J-M, Huang H, St Lezin E, Spence MA, Flodman P, Printz M, Lathrop GM, Vergnaud G, Kurtz TW. Mapping of a quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains. J Clin Invest. 1995;96:1973-1978.
Bonnardeaux A, Nadaud S, Charru A, Jeunemaitre X, Corvol P, Soubrier F. Lack of evidence for linkage of endothelial cell nitric oxide synthase gene to essential hypertension. Circulation. 1995;91:96-102.
Rapp JP, Deng AY. Detection and positional cloning of blood pressure quantitative trait loci: is it possible? Hypertension. 1995;25:1121-1128.
Jacob HJ, Brown DM, Bunder RK, Daly MJ, Dzau VJ, Goodman A, Koike G, Kren V, Kurtz T, Lernmark A, Levan G, Mao Y-P, Pettersson A, Pravenec M, Simon JS, Szpirer C, Szpirer J, Trolliet MR, Winer ES, Lander ES. A genetic linkage map of the laboratory rat, Rattus norvegicus. Nat Genet. 1995;9:63-69.
Serikawa T, Kuramoto T, Hilbert P, Mori M, Yamada J, Dubay CJ, Lindpaintner K, Ganten D, Guénet J-L, Lathrop GM, Beckmann JS. Rat gene mapping using PCR-analyzed microsatellites. Genetics. 1992;131:701-721.
Laubach VE, Shesely EG, Smithies O, Sherman PA. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci U S A. 1995;92:10688-10692.
Ikeda Y, Saito K, Kim J-I, Yokoyama M. Nitric oxide synthase isoform activities in kidney of Dahl salt-sensitive rats. Hypertension. 1995;26(part 2):1030-1034.