Baroreflex control of heart rate in young and adult salt hypertensive inbred Dahl rats

Baroreflex control of heart rate was studied in inbred salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) Dahl rats that were subjected to chronic dietary sodium chloride loading (for 4 weeks) either in youth or only in adulthood, i.e. from the age of 4 or 12 weeks. Using phenylephrine administration to pentobarbital-anesthetized male rats we have demonstrated the decreased baroreflex sensitivity (lower slope for reflex bradycardia) in young prehypertensive SS/Jr rats fed a low-salt diet as compared to age-matched SR/Jr animals. High salt intake further suppressed baroreflex sensitivity in young SS/Jr but not in SR/Jr rats. Baroreflex sensitivity decreased with age in SR/Jr rats, whereas it increased in SS/Jr rats fed a low-salt diet. Thus at the age of 16 weeks baroreflex sensitivity was much higher in SS/Jr than in SR/Jr animals. High salt intake lowered baroreflex sensitivity even in adult SS/Jr rats without affecting it in adult SR/Jr rats. Nevertheless, baroreflex sensitivity was significantly lower in young SS/Jr rats with a severe salt hypertension than in adult ones with a moderate blood pressure elevation. It is concluded that the alterations of baroreflex sensitivity in young inbred SS/Jr rats (including the response to high salt intake) are similar to those described earlier for outbred salt-sensitive Dahl rats. We have, however, disclosed contrasting age-dependent changes of baroreflex sensitivity in both inbred substrains of Dahl rats.     

Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?

There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3 h of reperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection.     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

Cadmium,iron and zinc interaction and hematological parameters in rat dams and their offspring

The effects of cadmium (Cd) were evaluated in offspring exposed from birth until weaning (neonatal day 0–21) and 4 weeks after exposure cessation focusing on iron (Fe) and zinc (Zn) levels in organs and hematological parameters. Wistar female rats were administered 50 mg Cd/L in drinking water (Cd-exposed) for 4 weeks before mating and during 3 weeks of gestation plus 3 weeks of lactation. Controls were supplied drinking water. At birth, part of Cd-exposed dams’ litters was cross-fostered to control dams (CCd group) and their control litters were cross-fostered to Cd-exposed dams (CdC group). This procedure enabled to discern the effects of gestational, lactational and gestational plus lactational Cd exposure until weaning in F₁ offspring. Elements were analyzed by atomic absorption spectrometry; hematological parameters manually; and histopathological changes by light microscopy. Gestational plus lactational exposure in Cd-exposed dams and their offspring increased Cd and decreased Fe levels, increased Zn in dams and decreased Zn and body weights in 11- and 21-day pups. In 21-day weanling pups, decreased red blood cell (RBC) count, hemoglobin and hematocrit values and increased reticulocytes in peripheral blood were also found with concomitant histopathological finding of extramedullary hematopoiesis in the liver. In cross-fostered pups with gestational exposure (CCd pups), Fe in the liver decreased on day 11 and Zn increased in the kidney on day 21 whereas in pups with lactational exposure (CdC pups) Zn in the brain decreased on day 11 and Fe decreased in the liver and brain on day 21. Regardless of exposure cessation at weaning, in offspring with gestational plus lactational exposure (Cd-exposed) body weights, kidney and brain Fe levels and RBC and hemoglobin remained decreased in blood until puberty. Furthermore Zn levels increased in the liver, kidney and brain. It was concluded that gestational plus lactational Cd exposure caused decreases in Fe and Zn levels and hematotoxic effects in F₁ offspring more pronouncedly than exposure during either gestational or lactational period alone and the adverse effects of maternally mediated Cd exposure continued after exposure cessation into adulthood.     

Stimulation of brain Na+ channels by FMRFamide in Dahl SS and SR rats

Stimulation of brain Na+ channels by Phe-Met-Arg-Phe-NH2 (FMRFamide) increases sympathetic nerve activity and blood pressure (BP) in Wistar rats. Blockade of brain ouabain-like compounds (OLC) by specific antibody Fab fragments prevents these responses to intracerebroventricular FMRFamide. In the present study, we evaluated the effects of high-salt intake on brain FMRFamide levels and the responses of BP and brain OLC to intracerebroventricular infusion of FMRFamide in Dahl salt-sensitive (SS) and salt-resistant (SR) rats. FMRFamide and OLC content was measured with the use of RIA and ELISA, respectively. A high-salt diet (1,370 micromol Na+/g) for 2 wk significantly increased BP in Dahl SS but not in SR rats. On a regular salt diet, Dahl SS and SR rats showed similar FMRFamide levels in the whole hypothalamus, pons and medulla, and spinal cord. A high-salt diet for 2 wk did not affect FMRFamide levels in these tissues in both Dahl SS and SR rats. In Dahl SS but not in SR rats, chronic intracerebroventricular infusion of FMRFamide (200 nmol. kg(-1).day(-1)) for 2 wk significantly increased BP (mean arterial pressure: 116 +/- 5 vs. 100 +/- 2 mmHg; P < 0.01). Chronic intracerebroventricular infusion of FMRFamide significantly increased hypothalamic and pituitary OLC in Dahl SS but not SR rats. These results indicate that Dahl SS rats exhibit enhanced central responses to FMRFamide. In Dahl SS but not in SR rats on a high-salt diet, enhanced Na+ entry through FMRFamide-activated brain Na+ channels may increase brain OLC release, thereby leading to hypertension.     

High salt differentially regulates surface NKCC2 expression in thick ascending limbs of Dahl salt-sensitive and salt-resistant rats

NaCl reabsorption by the thick ascending limb of the loop of Henle (THAL) occurs via the apical Na-K-2Cl cotransporter, NKCC2. Overall, NKCC2 activity and NaCl reabsorption are regulated by the amount of NKCC2 at the apical surface, and also by phosphorylation. Dahl salt-sensitive rats (SS) exhibit higher NaCl reabsorption by the THAL compared with Dahl salt-resistant rats (SR), and they become hypertensive during high-salt (HS) intake. However, the effect of HS on THAL transport, surface NKCC2 expression, and NKCC2 NH(2)-terminus phosphorylation has not been studied. We hypothesized that HS enhances surface NKCC2 and its phosphorylation in THALs from Dahl SS. THAL suspensions were obtained from a group of SS and SR rats on normal-salt (NS) or HS intake. In SR rats THAL NaCl transport measured as furosemide-sensitive oxygen consumption was decreased by HS (-34%, P < 0.05). In contrast, HS did not affect THAL transport in SS rats. As expected, HS increased systolic blood pressure only in SS rats (Δ 23 ± 2 mmHg, P < 0.002) but not in SR rats (Δ 5 ± 3 mmHg). We next tested the effect of HS intake on apical surface NKCC2 and its NH(2)-terminus threonine phosphorylation (P-NKCC2) in SS and SR rats. HS intake decreased surface NKCC2 by 15 ± 2% (P < 0.03) in THALs from SR without affecting total NKCC2 or NH(2)-terminus P-NKCC2. In contrast, in SS rats HS intake increased surface NKCC2 by 54 ± 6% (P < 0.01) without affecting total NKCC2 expression or P-NKCC2. We conclude that HS intake causes different effects on surface NKCC2 in SS and SR rats. Our data suggest that enhanced surface NKCC2 in SS rats might contribute to enhanced NaCl reabsorption in SS rats during HS intake.     

Role of the adenosine(2A) receptor-epoxyeicosatrienoic acid pathway in the development of salt-sensitive hypertension

Activation of rat adenosine(2A) receptors (A(2A) R) dilates preglomerular microvessels, an effect mediated by epoxyeicosatrienoic acids (EETs). High salt (HS) intake increases epoxygenase activity and adenosine levels. A greater vasodilator response to a stable adenosine analog, 2-chloroadenosine (2-CA), was seen in kidneys obtained from HS-fed rats which was mediated by increased EET release. Because this pathway is antipressor, we examined the role of the A(2A) R-EET pathway in a genetic model of salt-sensitive hypertension, the Dahl salt-sensitive (SS) rats. Dahl salt resistant (SR) rats fed a HS diet demonstrated a greater renal vasodilator response to 2-CA. In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed normal salt (NS) or HS diet. In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A) R and cytochrome P450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake. In vivo inhibition of the A(2A) R-EET pathway in Dahl SR rats fed a HS diet results in reduced renal EETs levels, diminished natriuretic capacity and hypertension, thus supporting a role for the A(2A) R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading. An inability of Dahl SS rats to upregulate the A(2A) R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.     

Four polymorphic markers on rat chromosome 12 form a single linkage group

Four PCR-typable polymorphic markers were mapped to rat chromosome 12 by linkage analysis of F₂ intercross progeny of Fischer (F344/N) and Lewis (LEW/N) rat strains. The markers formed a single linkage group, covering 27.7 cM, with the following order and distance between markers: plasminogen activator inhibitor (Planh)—0.0 cM—phosphoenolpyruvate carboxykinase-related sequence 2 (Pepckr2)—15.4 cM—anonymous marker (D12N155)—12.3 cM—serine dehydratase (Sdh). All markers were identified and genotyped by PCR analysis of simple sequence repeats. The gene encoding Planh was previously assigned to rat chromosome 12, which allowed us to assign the entire linkage group to this chromosome. These markers were highly polymorphic in 13 additional inbred rat strains (BUF/N, BN/SsN, WKY/N, MNR/N, LER/N, WBB1/N, WBB2/N, MR/N, LOU/MN, SHR/N, ACI/N, SR/Jr, and SS/Jr). These markers should be useful tools for further genetic studies in rats.     

Coexisting independent sodium-sensitive and sodium-insensitive mechanisms of genetic hypertension in spontaneously hypertensive rats (SHR)

Some essential hypertensive patients and genetic hypertensive rat strains have less than the normal levels of Mg2+ tightly bound to the plasma membranes of their erythrocytes and other cells, i.e., the magnesium binding defect (MgBD). This binding defect appears to cause increased passive permeability of the membrane to Na+ and thereby its increased intracellular concentration, particularly if the Na+-extrusion enzyme systems of the cell are also defective. The Na+-Ca2+ exchange system in the cell membrane exports Na+ and imports Ca2+, increasing the tone of the smooth muscle cell and thus producing hypertension (HTn). This HTn is Na+-sensitive. Evidence supporting this postulate was obtained by determining the intraerythrocyte total concentrations of Na+, Ca2+, K+, and Mg2+ in two strains of spontaneously hypertensive rats (SHR and SS/Jr rats, having the MgBD together with the other requisites of the Na+-sensitive pathway) and their respective controls (WKY and SR/Jr rats, in which this complete pathway is absent). The Na+ and Ca2+ concentrations in the hypertensive rats were increased, and that of K+ was decreased. The concentrations of these cations were very similar in the two hypertensive strains. The level of membrane tightly bound Ca2+ in SHR erythrocyte membranes was significantly higher than those in the other three rat strains, which were not statistically different from each other. These results support previously reported evidence of the existence of a novel HTn-generating mechanism in the SHR rat, in which the intracellular Ca2+ concentration is increased as the result of the enhanced diffusion of this ion into the cell and the accompanying deficiency of the Ca2+ extrusion enzyme systems. This pathway is therefore Na+-insensitive, i.e., Ca2+-sensitive.     

The Impact of Maternal Uterine Genotype on Postnatal Growth and Adult Body Size in Mice

Embryo transfers were used to demonstrate that the genotype of the mother providing the uterine developmental environment significantly influences postnatal growth and adult body size of her progeny. Irrespective of their own genotype, mouse embryos transferred into the uterus of an inbred strain with large body size (C3H) had greater body weights, longer tails and higher growth rates than those transferred into the uterus of a strain with small body size (SWR). Uterine heterosis on body size was smaller than progeny heterosis, and both progeny and uterine heterosis persisted in adult mice. Uterine litter size was significantly negatively associated with body weight, tail length, growth rate and the timing of developmental events. The inbred SWR strain was more sensitive to the embryo transfer procedure than the C3H strain, but effects due to embryo transfer were moderate. Prenatal uterine effects have ramifications for biotechnologies utilizing embryo transfer as well as predictions about evolutionary change by selection.     

Developmental arrest and pregnancy-induced transmammary transmission of Ancylostoma caninum larvae in the murine model

Pregnancy is associated with reactivation of latent infections of many protozoal and helminthic parasites. To facilitate in vivo studies on the process of transmammary transmission of hookworm infection to nursing newborns, we established an experimental model of infection of BALB/c mice with infective larvae of the canine nematode Ancylostoma caninum. To establish latency with a significant reservoir of tissue larvae and achieve acceptable pregnancy success rates, mice were subcutaneously infected at day 5 postimpregnation; similar larval distribution profiles were observed at the end of the gestational period for bred compared to correspondingly infected unbred animals. No larvae were detected in fetuses or neonatal pups. Significant numbers of larvae were not detected in mammary tissue during the periparturient or postpartum lactational periods although about 8% of a dam's reservoir of tissue larvae was transferred to her nursing pups; this suggests that larvae reaching the mammary glands are rapidly transmitted through the milk sinuses, as was documented by histopathological analyses. Comparison of BALB/c with C57BL/6 mice that typically display divergent immune responses to infection showed no difference in tissue larval burden or in numbers transferred to pups. A hypothesis for the molecular mechanism of larval reactivation and transmission is discussed.     

Catecholamines and atrial natriuretic factor in Dahl and spontaneously hypertensive rats

We have previously demonstrated two different catecholaminergic patterns in genetic and experimental hypertension: a hyperdopaminergic state in spontaneously hypertensive (Okamoto) rats (SHR) and a hypernoradrenergic state in salt-sensitive Dahl rats. Plasma immunoreactive atrial natriuretic factor (IR ANF) concentrations increase in both models as a response to hypertension. To distinguish between the genetic and acquired components of these abnormalities, we measured adrenal dopamine-beta-hydroxylase (D beta H) activity and coeliac ganglionic atrial natriuretic factor (ANF) like immunoreactivity in the two animal strains. While adrenal D beta H activity was increased in Dahl S rats, it was diminished in SHR in the prehypertensive as well as in the hypertensive stages. In the hypertensive stage, the ANF-like immunoreactivity in the coeliac ganglia was lower in the Dahl S group but higher in SHR than in their respective normotensive controls; there were no changes in these animals when they were prehypertensive. Differences in D beta H activity, which determines the fine tuning of sympathoadrenomedullary catecholamine synthesis may account for the inheritance of mechanisms resulting in salt-sensitive hypertension (as in SHR) or salt-dependent hypertension (as in Dahl salt-sensitive rats). In contrast, plasma IR ANF concentrations may reflect a defense mechanism against hypertension. However ANF-like immunoreactivity in coeliac ganglia does not follow its plasma concentrations and changes in different directions in the two hypertensive strains; it may reflect a neuromodulatory function of ANF in the ganglionic neurotransmission and different implications of this role of ANF in the two hypertensive models.     

Influence of exercise training on resting blood pressures of Dahl rats

To determine whether female Dahl salt-sensitive (SS) hypertensive rats would adapt to chronic treadmill exercise by exhibiting lower resting systolic blood pressures (RSBP), a 12-wk training program was undertaken. Female Dahl salt-resistant (SR) rats were also trained for the same time period a a similar intensity [40-70% maximal O2 consumption (VO2max)] and duration (55 min). Postexperimental treadmill run times and VO2max values [SR: nontrained (NT) 87 +/- 1, trained (T) 97 +/- 2; SS: NT 82 +/- 2, T 92 +/- 3 ml.min-1 X min-1 X kg-1] indicated that the prescribed program had produced a trained state. However, the training program caused no group differences between the SR or the SS and their nontrained controls in measurements associated with sodium chloride intake, fluid consumption, urine production, 24-h sodium excretion, plasma volumes, plasma insulin, or blood volumes. Chronic exercise did significantly lower RSBP in the SR subgroup after 6 wk (NT 123 +/- 4, T 110 +/- 3 mmHg) and 8 wk (NT 120 +/- 4, T 106 +/- 2 mmHg) and remained lower throughout the remaining weeks of the experiment. On the other hand, the RSBP results of the trained SS rats were significantly higher than the nontrained SS rats after 6 wk (NT 155 +/- 8, T 191 +/- 7 mmHg) and were never significantly different than the controls for the remainder of the study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex-specific differences in chromosome-dependent regulation of vascular reactivity in female consomic rat strains from a SSxBN cross

High-throughput studies in the Medical College of Wisconsin Program for Genomic Applications (Physgen) were designed to link chromosomes with physiological function in consomic strains derived from a cross between Dahl salt-sensitive SS/JrHsdMcwi (SS) and Brown Norway normotensive BN/NHsdMcwi (BN) rats. The specific goal of the vascular protocol was to characterize the responses of aortic rings from these strains to vasoconstrictor and vasodilator stimuli (phenylephrine, acetylcholine, sodium nitroprusside, and bath hypoxia) to identify chromosomes that either increase or decrease vascular reactivity to these vasoactive stimuli. Because previous studies demonstrated sex-specific quantitative trait loci (QTLs) related to regulation of cardiovascular phenotypes in an F2 cross between the parental strains, males and females of each consomic strain were included in all experiments. As there were significant sex-specific differences in aortic sensitivity to vasoconstrictor and vasodilator stimuli compared with the parental SS strain, we report the results of the females separately from the males. There were also sex-specific differences in aortic ring sensitivity to these vasoactive stimuli in consomic strains that were fed a high-salt diet (4% NaCl) for 3 wk to evaluate salt-induced changes in vascular reactivity. Differences in genetic architecture could contribute to sex-specific differences in the development and expression of cardiovascular diseases via differential regulation and expression of genes. Our findings are the first to link physiological traits with specific chromosomes in female SS rats and support the idea that sex is an important environmental variable that plays a role in the expression and regulation of genes.     

Whole-Genome Sequences of DA and F344 Rats with Different Susceptibilities to Arthritis,Autoimmunity, Inflammation and Cancer

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease.     

Potassium turnover and norepinephrine sensitivity in the thoracic aorta of the Dahl rat

This in vitro study evaluated the basal 42K turnover and response to norepinephrine (NE) in the thoracic aorta removed from Dahl salt-sensitive (S) and salt-resistant (R) rats. Five-week-old S and R rats were placed on either a high-salt (HS) or low-salt (LS) diet. After 5 weeks of the diet, systolic blood pressure, aortic weight/length ratio, and the cellular pool of K+ were elevated in the S-HS group only. In contrast, the steady state turnover of 42K, the NE ED50, and the response to a supramaximal dose of NE were the same in both groups of salt-sensitive and salt-resistant rats. These results suggest that, despite the presence of a greatly elevated systolic blood pressure and evidence of aortic hypertrophy, the intrinsic electrolyte metabolism of the vascular smooth muscle in the Dahl hypertensive rat is the same as that of the Dahl normotensive rat.     

Transient hypercapnic stress causes exaggerated and prolonged elevation of cardiac and renal interstitial norepinephrine levels in conscious hypertensive rats

The responses of sympathetic nerve activity to transient stress can be exaggerated in salt-sensitive (SS), hypertensive subjects. Cardiac and renal interstitial norepinephrine (iNE) levels during and after transient hypercapnia were investigated in conscious SS rats. Dahl SS and salt-resistant (SR) 6-wk-old rats were fed a high-salt diet, and at 12 wk iNE levels in the heart and kidney were determined using microdialysis with probes inserted in the left ventricular (LV) wall and kidney. A telemetry system determined blood pressure and heart rate (HR) in separate animals. After recovery from the operation, data were collected before, during, and after exposure to normoxic 10% CO(2) for 25 min under unanesthetized conditions. The plasma NE concentrations at baseline did not differ between the two strains. Both cardiac and renal iNE levels were much higher in SS rats than in SR rats at baseline as well as during hypercapnic stress. After stress, the markedly increased iNE levels of SS rats were prolonged in the LV as well as in the kidney. During hypercapnic stress, HR decreased in both SS and SR rats, while sudden increases in HR immediately after the withdrawal from stress were followed by its slower reduction in SS rats compared with SR rats. In conclusion, transient hypercapnic stress causes exaggerated and prolonged elevation of iNE levels in the heart as well as in kidneys of SS animals.