Genetic stress-reactivity, sex, and conditioning intensity affect stress-enhanced fear learning.

The Stress-Enhanced Fear Learning (SEFL) model of posttraumatic stress disorder (PTSD) reveals increased fear memory in animals exposed to stress prior to contextual fear conditioning (CFC), similar to the increased likelihood of developing PTSD in humans after prior stress. The present study utilized the SEFL model by exposing animals to restraint stress as the first stressor, followed by CFC using foot-shocks with 0.6 mA or 0.8 mA intensity. Adult males and females from the two nearly isogenic rat strains, the genetically more stress-reactive Wistar Kyoto (WKY) More Immobile (WMI), and the less stress-reactive WKY Less Immobile (WLI) were employed. Percent time spent freezing at acquisition and at recall differed between these strains in both prior stress and no stress conditions. The significant correlations between percent freezing at acquisition and at recall suggest that fear memory differences represent a true phenotype related to the stress-reactivity differences between the strains. This assumption is further substantiated by the lack of effect of either conditioning intensity on percent freezing in WLI males, while WMI males were affected by both intensities albeit with opposite directional changes after prior stress. Differences between the sexes in sensitivity to the two conditioning intensities became apparent by the opposite directional and inverse relationship between fear memory and the intensity of conditioning in WMI males and females. The present data also illustrate that although corticosterone (CORT) responses to prior stress are known to be necessary for SEFL, plasma CORT and percent freezing were positively correlated only in the stress less-reactive WLI strain. These differences in baseline fear acquisition, fear memory, and the percent freezing responses to the SEFL paradigm in the two genetically close inbred WMI and WLI strains provide a unique opportunity to study the genetic contribution to the variation in these phenotypes.

Methylphenidate and Atomoxetine-Responsive Prefrontal Cortical Genetic Overlaps in "Impulsive" SHR/NCrl and Wistar Rats.

Impulsivity, the predisposition to act prematurely without foresight, is associated with a number of neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). Identifying genetic underpinnings of impulsive behavior may help decipher the complex etiology and neurobiological factors of disorders marked by impulsivity. To identify potential genetic factors of impulsivity, we examined common differentially expressed genes (DEGs) in the prefrontal cortex (PFC) of adolescent SHR/NCrl and Wistar rats, which showed marked decrease in preference for the large but delayed reward, compared with WKY/NCrl rats, in the delay discounting task. Of these DEGs, we examined drug-responsive transcripts whose mRNA levels were altered following treatment (in SHR/NCrl and Wistar rats) with drugs that alleviate impulsivity, namely, the ADHD medications methylphenidate and atomoxetine. Prefrontal cortical genetic overlaps between SHR/NCrl and Wistar rats in comparison with WKY/NCrl included genes associated with transcription (e.g., Btg2, Fos, Nr4a2), synaptic plasticity (e.g., Arc, Homer2), and neuron apoptosis (Grik2, Nmnat1). Treatment with methylphenidate and/or atomoxetine increased choice of the large, delayed reward in SHR/NCrl and Wistar rats and changed, in varying degrees, mRNA levels of Nr4a2, Btg2, and Homer2, genes with previously described roles in neuropsychiatric disorders characterized by impulsivity. While further studies are required, we dissected potential genetic factors that may influence impulsivity by identifying genetic overlaps in the PFC of "impulsive" SHR/NCrl and Wistar rats. Notably, these are also drug-responsive transcripts which may be studied further as biomarkers to predict response to ADHD drugs, and as potential targets for the development of treatments to improve impulsivity.

SHRSP/Izm and WKY/NCrlCrlj rats having a missense mutation in Abcg5 deposited plant sterols in the body, but did not change their biliary secretion and lymphatic absorption-comparison with Jcl:Wistar and WKY/Izm rats.

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.

Test- and behavior-specific genetic factors affect WKY hypoactivity in tests of emotionality.

Inbred Wistar-Kyoto rats consistently display hypoactivity in tests of emotional behavior. We used them to test the hypothesis that the genetic factors underlying the behavioral decision-making process will vary in different environmental contexts. The contexts used were the open-field test (OFT), a novel environment with no explicit threats present, and the defensive-burying test (DB), a habituated environment into which a threat has been introduced. Rearing, a voluntary behavior was measured in both tests, and our study was the first to look for genetic loci affecting grooming, a relatively automatic, stress-responsive stereotyped behavior. Quantitative trait locus analysis was performed on a population of 486 F2 animals bred from reciprocal inter-crosses. The genetic architectures of DB and OFT rearing, and of DB and OFT grooming, were compared. There were no common loci affecting grooming behavior in both tests. These different contexts produced the stereotyped behavior via different pathways, and genetic factors seem to influence the decision-making pathways and not the expression of the behavior. Three loci were found that affected rearing behavior in both tests. However, in both contexts, other loci had greater effects on the behavior. Our results imply that environmental context's effects on decision-making vary depending on the category of behavior.

Quantitative trait loci associated with elevated thyroid-stimulating hormone in the Wistar-Kyoto rat.

Thyroid hormones are essential for the regulation of developmental and physiological processes. The genetic factors underlying naturally occurring variability in mammalian thyroid function are, however, only partially understood. Genetic control of thyroid function can be studied with animal models such as the inbred Wistar-Kyoto (WKY) rat strain. Previous studies established that WKY rats have elevated TSH, slightly elevated total T3, and normal total T4 levels compared with Wistar controls. The present study confirmed a persistent 24-h elevation of TSH in WKY rats compared with the Fisher 344 (F344) rat, another inbred strain. Acute T3 challenge (25 microg/100 g body weight ip) suppressed serum TSH and T4 levels in both strains. Quantitative trait locus analysis of elevated TSH in a reciprocally bred WKY x F344 F2 population identified one highly significant locus on chromosome 6 (LOD=11.7, TSH-1) and one suggestive locus on chromosome 5 (LOD=2.3, TSH-2). The confidence interval of TSH-1 contains the TSH receptor and type 2 deiodinase genes, and TSH-2 contains the type 1 deiodinase gene. The WKY alleles of each gene contain sequence alterations, but additional studies are indicated to identify the specific gene or genes responsible for altered regulation of the thyroid axis. These findings suggest that one or more genetic alterations within the TSH-1 locus significantly contribute to the altered thyroid function tests of the WKY rat.

No involvement of the nerve growth factor gene locus in hypertension in spontaneously hypertensive rats.

Sympathetic hyper-innervation and increased levels of nerve growth factor (NGF), an essential neurotrophic factor for sympathetic neurons, have been observed in the vascular tissues of spontaneously hypertensive rats (SHRs). Such observations have suggested that the pathogenesis of hypertension might involve a qualitative or quantitative abnormality in the NGF protein, resulting from a significant mutation in the gene's promoter or coding region. In the present study, we analyzed the nucleotide sequences of the cis-element of the NGF gene in SHRs, stroke-prone SHRs (SHRSPs), and normotensive Wistar-Kyoto (WKY) rats. The present analyses revealed some differences in the 3-kb promoter region, coding exon, and 3' untranslated region (3'UTR) for the NGF gene among those strains. However, the observed differences did not lead to changes in promoter activity or to amino acid substitution; nor did they represent a link between the 3'UTR mutation of SHRSPs and elevated blood pressure in an F2 generation produced by crossbreeding SHRSPs with WKY rats. These results suggest that the NGF gene locus is not involved in hypertension in SHR/ SHRSP strains. The present study also revealed two differences between SHRs and WKY rats, as found in cultured vascular smooth muscle cells and in mRNA prepared from each strain. First, SHRs had higher expression levels of c-fos and c-jun genes, which encode the component of the AP-1 transcription factor that activates NGF gene transcription. Second, NGF mRNAs prepared from SHRs had a longer 3'UTR than those prepared from WKY rats. Although it remains to be determined whether these events play a role in the hypertension of SHR/SHRSP strains, the present results emphasize the importance of actively searching for aberrant trans-acting factor(s) leading to the enhanced expression of the NGF gene and NGF protein in SHR/SHRSP strains.

Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats.

In the present study we investigated the regulation of tyrosine hydroxylase (TH) by angiotensin II (Ang II) in an attempt to provide cellular and molecular evidence that this hormone has increased neuromodulatory actions in the spontaneously hypertensive (SH) rat brain. Neuronal cells in primary culture from the hypothalamus-brain stem of both normotensive [Wistar-Kyoto (WKY)] and SH rats have been used. These cultures mimic in vivo situations. Ang II caused a time-dependent increase in TH activity in WKY rat brain neurons. A maximal increase of 2.5-fold was observed with 100 nM Ang II in an actinomycin- and cycloheximide-dependent process. In addition, Ang II caused a parallel increase in TH messenger RNA (mRNA) levels, with a maximal stimulation of 5-fold in 4 h by 100 nM Ang II in WKY rat brain neurons. The stimulation of TH mRNA was mediated by the AT1 receptor subtype, resulted from an increase in its transcription, and involved activation of phospholipase C and protein kinase C. Antisense oligonucleotide for c-fos attenuated Ang II stimulation of TH mRNA in a time- and dose-dependent fashion, indicating an involvement of c-fos as a putative third messenger in Ang II stimulation of TH. Ang II also caused stimulation of TH activity and its mRNA levels in neuronal cultures of SH rat brain by a mechanism similar to that observed for neuronal cultures of WKY rat brain, involving AT1 receptors, protein kinase C, and c-fos. However, the stimulation of TH activity and that of TH mRNA were approximately 30% and 80% higher, respectively, in the SH rat brain neurons than those in the WKY rat brain neurons. In vivo experiments have been carried out to validate the elevated response of TH gene expression to Ang II in SH rat brain neuronal cultures. Ang II stimulated both TH activity and TH mRNA levels in the hypothalami and brain stems of adult WKY and SH rats. The level of stimulation in the brain of the SH rat was significantly higher than that in the WKY rat. These observations are consistent with an increase in AT1, receptor gene expression and suggest that increased TH gene expression could be the cellular/molecular basis for the greater neuromodulatory action of Ang II in the SH rat brain.

Genetic divergence between the Wistar-Kyoto rat and the spontaneously hypertensive rat.

A method of restriction fragment length polymorphism (RFLP) analysis was used to estimate the amount of genetic divergence between the spontaneously hypertensive rat (SHR) strain and the Wistar-Kyoto (WKY) strain. DNA from each strain was digested with eight restriction endonucleases and hybridized with six single copy gene sequences. The number of hybridization bands in each digestion was used to estimate the total number of bases analyzed and RFLPs were scored as single mutations. Divergence was then estimated by dividing the number of mutations by the number of bases analyzed. In a total of 808 bases analyzed in WKY rats, a minimum of 13 mutations were scored in SHR, which yields a nucleotide divergence of 1 change per 62 bp. This is an extremely high amount of divergence given the known origin of these two strains and is comparable to the maximum divergence possible between unrelated humans.

Genetic heterogeneity of the spontaneously hypertensive rat.

We examined DNA fingerprints of the spontaneously hypertensive rat from Shimane Institute of Health Science, Izumo, Japan, including seven substrains that were separated in the early stages of the establishment of the stroke-prone spontaneously hypertensive rat, and compared their fingerprints with those of rats from other sources. Obtained DNA fingerprints revealed that, in both the stroke-resistant spontaneously hypertensive rat and the Wistar-Kyoto rat, there is a substantial genetic difference between the rats from the National Institutes of health and from Shimane Institute of Health Science. By contrast, only a small genetic difference was observed either between the rats from the National Institutes of Health and Charles River Laboratories or among the substrains of the spontaneously hypertensive rat in the Shimane Institute of Health Science. Further, in the strains from the Shimane Institute of Health Science, there were fingerprinting bands that could distinguish either the Wistar-Kyoto rat from all the substrains of the spontaneously hypertensive rat or the stroke-prone from the stroke-resistant spontaneously hypertensive rat in spite of their close genetic backgrounds. From the observations above, we concluded 1) that there is substantial genetic variance of the spontaneously hypertensive rat between the two major sources in the world, the National Institutes of Health and the Shimane Institute of Health Science and 2) that by DNA fingerprinting analysis, it is possible to identify the restriction fragment length polymorphisms that are specific for the spontaneously hypertensive rat or the stroke-prone spontaneously hypertensive rat. These polymorphisms can be applied in the segregation study of the F2 generation.

Molecular evidence of genetic heterogeneity in Wistar-Kyoto rats: implications for research with the spontaneously hypertensive rat.

Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto ("WKY") rats are frequently employed in experimental studies of hypertension. Although both SHR and WKY rat strains have been presumed to be fully inbred, recent studies have revealed important biologic variability in WKY rats from different commercial sources. Genealogic evidence suggests that, in the United States, breeding stocks of WKY rats may have been distributed to major commercial suppliers as early as the F10 generation. To test the hypothesis that commercially available WKY rats are genetically heterogeneous, we performed deoxyribonucleic acid (DNA) "fingerprint" analysis on genomic DNA of WKY rats from two of the largest vendors in the United States, Taconic Farms and Charles River Laboratories. We found molecular evidence of genetic variability not only among WKY rats from two different breeding facilities, but also among WKY rats within a single breeding facility (Taconic Farms). Although some studies have suggested the possibility of biologic variability in SHR from different sources, preliminary studies have not revealed molecular evidence of genetic heterogeneity in SHR from these vendors. In demonstrating genetic variability in WKY rats from different sources, the current study provides compelling evidence that rats designated WKY do not constitute an inbred strain. Accordingly, the results of studies in which SHR and WKY rats are compared might vary because of genetic heterogeneity in "the WKY rat control strain."

Hypertensive strains and normotensive 'control' strains. How closely are they related?

The spontaneously hypertensive rat and the Dahl salt-sensitive rat are the most widely studied genetic models of hypertension. Many investigators have attempted to study the pathogenesis of hypertension by comparing these strains with their respective normotensive "controls," the Wistar-Kyoto rat and the Dahl salt-resistant rat. However, the genetic relation between each of these hypertensive strains and its corresponding normotensive control has never been clearly defined. Based on an analysis of DNA "fingerprint" patterns generated with six multilocus probes, we found that the spontaneously hypertensive rat (Charles River Laboratories, Inc.) is genetically quite different from its normotensive Wistar-Kyoto control: these strains only share approximately 50% of their DNA fingerprint bands in common. The inbred Dahl salt-sensitive rat (SS/Jr strain) (Harlan Sprague Dawley, Inc.) and the Dahl salt-resistant rat (SR/Jr strain) share approximately 80% of their DNA fingerprint bands in common. To the extent that the genes identified by DNA fingerprint analysis are representative of loci dispersed throughout the rodent genome, the current findings provide evidence of extensive genetic polymorphism between these commonly used hypertensive strains and their corresponding normotensive controls, particularly in the spontaneously hypertensive rat model. These findings, together with the fact that an enormous number of biochemical and physiological differences have been reported between these hypertensive and normotensive strains, suggest that continued comparison of spontaneously hypertensive rats with Wistar-Kyoto rats or Dahl salt-sensitive with salt-resistant rats will have limited value for investigating the pathogenesis of hypertension.

The hypertensive Y chromosome elevates blood pressure in F11 normotensive rats.

Our laboratory has shown that the Y chromosome has a significant effect on blood pressure in the spontaneously hypertensive rat (SHR) model of hypertension and that the testes and androgen receptor contribute to the blood pressure rise. As an extension of our research, we have developed two new rat strains, SHR/a and SHR/y (F11) to study the Y chromosome. The objectives of the following research were 1) to study the blood pressure of rats with an SHR Y chromosome in a normotensive genetic background (SHR/y) or a normotensive Y chromosome in an SHR genetic background (SHR/a), 2) to determine the effect of male sex phenotype on the blood pressure of these rats, 3) to determine if testosterone replacement in castrated rats would restore blood pressure, and 4) to determine whether the Y chromosome from the SHR/y strain when crossed with a normotensive female can induce hypertension in androgen receptor-deficient male offspring. Blood pressure of male SHR/y rats was significantly higher than that of normotensive Wistar-Kyoto males (p < 0.01), and SHR/a males had significantly lower blood pressure compared with that of the parent SHR strain (p = 0.05). Testosterone replacement in castrated rats of both strains (SHR/a and SHR/y) restored blood pressure to control levels. Normotensive female King-Holtzman rats heterozygous for the testicular feminization gene were crossed with F11 SHR/a and SHR/y males.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological variability in Wistar-Kyoto rats. Implications for research with the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) initially bred in Kyoto is the most widely studied animal model of essential hypertension. As controls for the SHR, most workers have used normotensive descendants of Wistar rats from the colony in Kyoto from which the SHR strain was derived (Wistar-Kyoto rats, WKY). But the presumption that WKY are serviceable controls for SHR rests on the tacit assumption that all WKY constitute a single inbred strain. It appears, however, that whereas the National Institutes of Health distributed breeding stocks of SHR after they had been fully inbred (i.e., after 20 generations of brother-sister mating), the breeding stocks of WKY were distributed before they had been fully inbred. Accordingly, the biological variability of WKY may be greater than that of SHR. To investigate this possibility, we obtained SHR and WKY from two of the largest commercial suppliers in the United States and systematically measured the growth rate and blood pressure of these rats under identical physical and metabolic conditions. We found that WKY from one source differed from those of the other in both growth rate and blood pressure. In contrast, the SHR from the two suppliers were not different with respect to either growth rate or blood pressure. Because the National Institutes of Health may have distributed breeding stocks of WKY as early as the F6 generation, it is possible that rats currently designated as WKY do not constitute a single inbred strain. Thus, interpretation of studies employing "the Wistar-Kyoto rat strain" as a control for the SHR may be much more problematic than has previously been recognized.

Cloning, characterization, and genetic mapping of the rat type 2 angiotensin II receptor gene.

The renin-angiotensin system plays an important role in blood pressure homeostasis, but the contribution of the type 2 angiotensin II receptor (AT2R) is still unclear. The reports that the AT2R gene has been mapped to the X chromosome in human and rat and the previous report of a gene, Bp3, on the X chromosome responsible for an increase in blood pressure have suggested that the rat AT2R gene (Agtr2) could be this gene. To elucidate whether Agtr2 is Bp3, Agtr2 was cloned. A simple sequence repeat in the 3'-flanking region of this gene was identified and used as a genetic marker to map Agtr2 to the X chromosome at 18.1 cM distal to the androgen receptor locus. This map position is outside the confidence interval reported for Bp3, demonstrating that Agtr2 cannot be Bp3. However, these data will enhance the research into the AT2R biology as well as the study of the X chromosome.

Cloning of the alpha-subunit of GS protein from spontaneously hypertensive rats.

Enhanced sodium reabsorption by the kidney has a significant role in the development of genetic hypertension. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, the enhanced sodium reabsorption likely arises from abnormal hormonal regulation of tubular transport. Since hormonal signaling pathways are coupled frequently via GTP binding proteins, one explanation for hormonal abnormalities in SHR would be a defect in a GTP binding protein or proteins. Recent work has suggested that the regulation of Na+,K(+)-ATPase activity by cholera toxin-sensitive GTP binding proteins is abnormal in SHR. The purpose of the present studies was to clone the alpha S-subunit, which is the subunit ADP ribosylated by cholera toxin, of GS protein to determine whether it is abnormal in SHR. Reverse transcription-polymerase chain reaction was able to detect mRNA for alpha S in both Wistar-Kyoto (WKY) rats and SHR. Northern analysis indicated that equivalent amounts of alpha S mRNA were present in WKY rats and SHR. S1 nuclease analysis demonstrated that there was no difference in the amount of alpha S short and long forms between WKY rats and SHR. Subcloning and sequencing of polymerase chain reaction products from WKY rats and SHR indicated that the alpha S forms present in renal cortex were identical. ADP ribosylation studies with cholera toxin demonstrated the presence of equivalent amounts of alpha S protein in WKY rats and SHR. Taken together, these results suggest that the abnormal regulation of Na+,K(+)-ATPase activity by a cholera toxin-sensitive pathway in SHR does not arise from a defect in the alpha S subunit.

N-acetylation of aromatic amines: genetic polymorphism in inbred rat strains.

The inheritance of rat liver N-acetyltransferase polymorphism was investigated with reciprocal genetic crosses between slow (NSD/N) and rapid (Peth/N) acetylator strains. Rat liver N-acetyltransferase activity was determined using a spectrophotometric assay which measured the amount of arylamine substrate present after incubation with N-acetyltransferase in vitro. Male N-acetyltransferase activities assayed in liver preparations using p-aminobenzoic acid and p-toluidine as substrates indicate bimodality of the parental strains and unimodality of the F-1 generation; limited data suggest trimodality (not significantly different from a 1:2:1 ratio) of the F-2 generation. Reciprocal crosses of WKY/N, another slow acetylator strain, and the Peth/N strain gave results similar to those of the NSD/N x Peth/N cross. Female N-acetyltransferase activities in all strains studied were lower than male N-acetyltransferase activities, but were similarly distributed in the parental and F-1 generations. The male/female N-acetyltransferase activity ratio was substrate- and genotype-dependent. Results show that regulation of the variation of rat liver N-acetyltransferase activity is consistent with autosomal Mendelian inheritance of two major alleles at a single gene locus.

Molecular genetics of the SA-gene: cosegregation with hypertension and mapping to rat chromosome 1.

OBJECTIVES: The SA-gene shows markedly higher levels of expression in the kidneys of spontaneously hypertensive rats (SHR) than in their non-hypertensive reference strain, the Wistar-Kyoto (WKY) rat. Based on the important role of the kidney in blood pressure regulation, the possibility has been raised that this gene, the translational product of which remains unknown, may participate in the pathogenesis of primary hypertension. The present study was conducted to test this hypothesis and to ascertain the chromosomal localization of the SA-gene. DESIGN: A cosegregation study was performed using an F2 intercross between stroke-prone SHR (SHRSP) and WKY rats, and a previously described restriction fragment length polymorphism of the SA-gene for characterization of genotype. Mapping of the SA-gene was accomplished by screening a somatic cell-hybrid panel and by linkage group analysis. RESULTS: A statistically significant difference in systolic blood pressure was found after sodium loading, but not under basal conditions between groups of rats defined by zygosity at the SA locus, consistent with a hypertensive effect of the SHRSP allele. No effect of SA genotype on diastolic blood pressure was observed. The SA-gene was localized on rat chromosome 1. CONCLUSIONS: This study establishes the SA locus on chromosome 1 as a region in which a gene or genes contributing to blood pressure regulation in this model are localized, and provides further evidence for a possible role of the SA-gene in the pathogenesis of hypertension.

A new genetic locus cosegregating with blood pressure in F2 progeny obtained from stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats.

BACKGROUND: Segregation studies using genomic polymorphisms on F2 progeny obtained from hypertensive rat models showed that a putative hypertensive gene is located close to the angiotensin converting enzyme (ACE) gene. However, it was suggested that additional major genes should contribute to the pathogenesis of hypertension. METHODS: F2 rats were obtained from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats of Izumo colony. Blood pressure was measured with a photoelectronic oscillometric tail-cuff method before and during salt loading. Genomic DNA was extracted from livers and digested with HaeIII or Rsal. DNA fingerprinting was performed with 26 32P-labelled human variable number of tandem repeats markers. RESULTS: Eighty-seven fingerprint bands polymorphic between SHRSP and WKY were obtained. When the distribution of these bands in the F2 progeny was studied, one fingerprint band (1/MCT96.1) showed a distorted distribution between the high- and low-blood pressure subpopulations of the F2 rats, suggesting that the band cosegregated with blood pressure. When blood pressure was compared between the F2 rats with [(+) rats] and without [(-) rats] the 1/MCT96.1 band, it was found that (-) rats had significantly higher basal and salt-loaded blood pressures than (+) rats. The 1/MCT96.1 locus was also shown to have no positive linkage with the ACE locus. CONCLUSION: The present study showed that examination of the allele distribution between subpopulations with extreme phenotype can be used in the screening of loci cosegregating with blood pressure. Furthermore, a locus not in the ACE region, showing cosegregation with blood pressure in F2 progeny from SHRSP and WKY rats, was found.

Structural alterations of the renin gene in stroke-prone spontaneously hypertensive rats: examination of genotype-phenotype correlations.

Primary hypertension is considered a polygenic, inherited disorder; to date, the nature of the genes involved remains unknown. In this study we present evidence for a structural difference in the gene coding for renin between the stroke-prone, spontaneously hypertensive rat (SHRSP) and its normotensive control, the Wistar-Kyoto rat (WKY). Restriction fragment analysis using hybridization against probes complementary to defined regions of the renin gene identified a deletion, approximately 700 base pair in size, within the first intron in SHRSP compared with WKY. This restriction fragment length polymorphism (RFLP) affects a part of the gene that is characterized by the presence of a multimeric tandem repeat element, where the occurrence of insertional/deletional events might be expected and have recently been shown in other rat strains. In order to test for a possible phenotypical representation of this RFLP, we studied a population (n = 115) of F2 hybrid rats derived from cross-breeding SHRSP with WKY. Using direct blood pressure measurements in conscious animals, we ruled out a cosegregation of systolic or diastolic blood pressure with renin genotype. Several other phenotypical parameters examined (heart rate, absolute and relative magnitude of changes in blood pressure induced by stress or dietary sodium loading, plasma renin activity, ventricular hypertrophy and tissue water content) also showed no cosegregation with genotype. Our findings are in contrast to a recently published study examining an RFLP of the renin gene distinguishing salt-sensitive and salt-resistant Dahl rats. Thus, cosegregation of genotype and phenotype are not consistent, although in both cases, structural differences in the same region of the renin gene separate the hypertensive strain from its normotensive controls. These data may suggest differential roles of the renin-angiotensin system in these two models of genetically predetermined hypertension.

Female Wistar-Kyoto and SHR/y rats have the same genotype but different patterns of expression of renin and angiotensinogen genes.

OBJECTIVES: To evaluate whether renin and angiotensinogen gene expression in females from two strains of rats that share the same autosomes and X chromosomes differs. Female SHR/y rats have the parental Wistar-Kyoto rat autosomes and X chromosomes and have no chromosomes of spontaneously hypertensive rat origin; thus they are genetically equivalent to female Wistar-Kyoto rats. DESIGN AND METHODS: Because these genes are regulated by steroid hormones, we investigated the effects of removal of estrogen (ovariectomy) and addition of androgen (testosterone implants) on three groups of female SHR/y rats and the parental rat strain Wistar-Kyoto rat with groups of intact (control) rats, rats subjected to ovariectomy at age 3 weeks, and rats subjected to ovariectomy with a testosterone implant at age 3 weeks. RESULTS: The combination of removing estrogen early in development and supplementing the ovariectomized females with testosterone revealed strain differences in response of blood pressure. Renin and angiotensinogen messenger RNA levels appear to be regulated coordinately within each strain, although actual levels of messenger RNA differ between the strains. CONCLUSIONS: Similar patterns of responses of renin and angiotensinogen genes to ovariectomy and ovariectomy plus testosterone suggest that regulation of the genes is likely to be similar or coordinate. Differences in regulation of renin-angiotensin system genes between strains may result from epigenetic mechanisms such as genome imprinting of these genes or of another gene that functions as a common regulator of renin and angiotensinogen.