Genetic influence on the renin-angiotensin system. Evidence for a renin inhibitor in hypertension-prone rats.

TWO STRAINS OF RATS WITH OPPOSITE GENETIC PROPENSITY FOR HYPERTENSION WERE TESTED FOR: (a) the sensitivity to injections of angiotensin and renin, and (b) the influence of their plasma on the reaction velocity of renin and its substrate in vitro. Intact hypertension-prone (S) rats on low salt had higher sensitivity to angiotensin and a lower sensitivity to renin than hypertension-resistant (R) rats. High NaCl diet did not change the response of the R rats to these injections, but increased the response to renin and angiotensin in intact S rats. Bilateral nephrectomy caused increased response to renin and a decreased response to angiotensin in the S rats, so that both strains were equivalent after bilateral nephrectomy. In vitro, plasma from intact S rats inhibited the activity of hog renin. Plasma from R rats showed no inhibition. The inhibitor disappeared after bilateral nephrectomy. It was speculated that renin inhibitor may be involved in the development of hypertension by increasing sensitivity to angiotensin and other hypertensinogenic stimuli.

Effects of adrenalectomy on blood pressure in salt-fed, hypertension-prone rats. Failure of hypertension to develop in absence of evidence of adrenal cortical tissue.

In adrenalectomized, genetically hypertension-prone rats, a high degree of correlation was found between evidence of functioning adrenal tissue and the development of salt hypertension. There is considerable evidence that some rats have the capacity to regenerate functioning adrenal cortical tissue from accessory glands and microscopic rests, sometimes in remote locations. Therefore, the criteria for continued absence of adrenal function after surgical adrenalectomy are critical. In this study we used three tests to validate the presence, or absence, of adrenal function: (a) a biochemical test, the quantitative, serial measurement of plasma glucocorticoids in individual rats; (b) a physiological test, the ability to survive a virtually sodium-free diet; and (c) the anatomical search for histological evidence of adrenal cortical tissue. Among those animals that developed hypertension after adrenalectomy, the correlation between plasma steroid concentration and blood pressure was statistically significant. We suspect that this correlation exists only during the period when cortical tissue is regenerating; it does not exist among intact animals with and without hypertension induced by salt. It was concluded that some adrenocortical function is necessary for salt hypertension to develop. The evidence was insufficient to settle the question whether the action of corticosteroids is causative, or whether they play a supporting, although necessary, role for an extraadrenal hypertensinogenic factor to become manifest.

Genetic influence on the development of renal hypertension in parabiotic rats. Evidence for a humoral factor.

The effects of several renal manipulations including uninephrectomy, unilateral renal artery constriction, and a combination of these two (Goldblatt procedure) were studied in two strains of rats with opposite constitutional predispositions to experimental hypertension. The protective value of intact renal tissue to protect against hypertension was shown to be genetically determined. The Goldblatt procedure carried out on only one member of a parabiotic pair induced hypertension in this operated rat but significant hypertension developed in the intact partner only when the operated animal belonged to the strain predisposed to hypertension. It was speculated that there were qualitative differences in the pressor signals of the two strains of rats. In the strain genetically predisposed to hypertension there are at least two pressor principles: (a) one which is common to both strains, not transmittable via the parabiosis junction and presumably related to the renin-angiotensin system; and (b) a second which is specific for the hypertension-prone strain and can be transmitted through the parabiosis junction. This transmittable agent is probably identical with the factor that produces salt hypertension and is associated with the salt-excreting mechanism.

Genetic influence on the development of renoprival hypertension in parabiotic rats. Evidence that a humoral hypertensinogenic factor is produced in kidney tissue of hypertension-prone rats.

Rats from two strains with opposite constitutional predisposition to hypertension were joined in parabiosis and one partner was nephrectomized. The influence of genetic factors and of diet on the blood pressures of the two classes of parabionts, operated and intact, indicated that renoprival hypertension occurred with equal frequency in rats from both strains; that the development of renoprival hypertension depended on the influence from an intact S partner, or on a high salt intake, or on both. A nephrectomized S rat developing renoprival hypertension did not induce high blood pressure in its intact R partner. In this respect renoprival hypertension differs from salt and renal hypertension. The findings are interpreted to mean that the hypertensinogenic agent specific for S rats is produced by S kidneys.

Effects of chronic excess salt ingestion. Inheritance of hypertension in the rat.

TWO STRAINS OF RAT HAVE BEEN DEVELOPED BY SELECTIVE BREEDING: one strain (R rats) is resistant to salt hypertension, the other strain (S rats) is highly susceptible. The inheritance of these traits has been explored in the first (F(1)) and second (F(2)) generation of crossbred rats and in backcrosses between parent and first filial (F(1) x R, F(1) x S) generations. Male F(1) rats had an average blood pressure close to the mid-parental (R and S) values, and the average of F(2) males was equivalent to that of F(1). Male offspring of F(1) with R, or F(1) with S also showed averages close to the respective mid-parental values. Female offspring showed deviations from this linear relationship, indicating a significant dominance in the female for the genes of normal blood pressure. A model of two autosomal, nonlinked diallelic loci, with a dominance deviation at one locus in the female, gave predictions with a reasonable agreement to the observed values. The same model also appeared compatible with human data if we assume a gene frequency of 0.13 for the hypertensinogenic allele on both loci. Random fluctuations in blood pressure, and incomplete homogeneity of parental strains permit several alternative models. The major conclusions are: that more than one locus is needed to explain the findings though as few as two loci may possibly suffice; the allelic effect seems additive in males, but there is a sex-determined influence on the expression in females; there is no consistent evidence for sex-linked inheritance. Furthermore, this model developed from the study of rats may provide a framework for analysis of human data.

Genetic predisposition and stress-induced hypertension.

When chronically exposed to an approach-avoidance conflict, rats with a genetic susceptibility to hypertension showed persisten elevations in systolic blood pressure, but rats with a genetic resistance to hypertension did not. Hence, psychic stress is selectively efficacious in producing hypertensive effects depending on genetic predisposition of the animal.

Regional myocardial substrate uptake in hypertensive rats: a quantitative autoradiographic measurement.

Severe hypertension causes global and regional changes in myocardial perfusion and substrate utilization. Regional perfusion and fatty acid utilization were evaluated by dual-tracer autoradiography in normotensive and hypertensive rats of the Dahl strain. The regional distributions of perfusion and fatty acid utilization were homogeneous in normotensive rats. Severe hypertension was associated with a homogeneous pattern of regional perfusion, but fatty acid utilization was focally decreased in the free wall of the left ventricle. The decrease in fatty acid uptake was associated with a concomitant increase in glucose utilization. These findings suggest that severe hypertension is associated with uniform myocardial perfusion and focal alterations in the substrates used for the performance of myocardial work.

Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo. An autocrine mechanism promoting hypertensive vascular hypertrophy.

To clarify the role of PDGF A-chain in hypertensive vascular hypertrophy of spontaneously hypertensive rats (SHRs), we studied levels of PDGF A-chain gene expression and transcription factors related to the gene in vascular smooth muscle cells (VSMCs) of SHRs in vivo. RNase protection assay and in situ hybridization showed that PDGF A-chain mRNA levels in VSMCs of SHRs were twofold higher than in those of normotensive Wistar-Kyoto rats. Gel retardation assays showed that levels of Sp1 and AP-2 in VSMCs of SHRs were twofold more abundant than in those of Wistar-Kyoto rats. Treatment with four pharmacologically different species of antihypertensive drugs for 2 wk decreased the levels of both PDGF A-chain mRNA and Sp1, but not AP-2 level in VSMCs of SHRs with regression of aortic hypertrophy, indicating that increases in levels of both PDGF A-chain mRNA and Sp1 in VSMCs of SHRs were associated with high blood pressure. These results suggest that high blood pressure is a stimulus which upregulates PDGF A-chain gene expression in VSMCs of SHRs, resulting in an autocrine enhancement in hypertensive vascular hypertrophy, and that the activation of the gene may be mediated through increases in Sp1 in these cells.

Expression of N-myc and c-src protooncogenes correlating to the undifferentiated phenotype and prognosis of primary neuroblastomas.

Genomic amplification of the N-myc protooncogene in neuroblastomas correctly predicts poor outcome for the patients. However, the prognosis for neuroblastomas with a single copy of N-myc is also poor in cases diagnosed after 1 year of age but good in infantile cases. To elucidate the different prognoses depending upon the age of the patients with neuroblastoma, we performed an analysis of the expression of protooncogenes related to neural differentiation. We examined the genomic amplification of N-myc in 26 specimens of neuroblastomas and further analyzed 22 of the 26 cases for expression of N-myc, c-src, c-Ha-ras, and c-fos. Consequently, we observed frequent overexpression of N-myc in undifferentiated neuroblastomas and enhanced expression of c-src and c-Ha-ras in infantile neuroblastomas with favorable prognosis and in neuroblastomas differentiated by chemotherapy. These findings suggest that c-src and c-Ha-ras play important roles in the neural differentiation of infantile neuroblastomas.

Identification of human DAN gene, mapping to the putative neuroblastoma tumor suppressor locus.

The expression of DAN gene (previously designated as N03 gene) is significantly reduced in a variety of transformed rat fibroblasts, including v-src- (SR-3Y1), SV40- and v-mos-transformed 3Y1 cells, compared with that in parental 3Y1 cells. Recently, DAN gene has been shown to possess a tumor suppressive activity when it is overexpressed in SR-3Y1 cells (Ozaki & Sakiyama, 1994). To assess the involvement of DAN gene with human neoplasms, we have isolated human DAN counterpart from a normal lung cDNA library by using rat DAN cDNA as a probe, and determined its chromosomal location. Human DAN gene mapped to chromosome 1p36.11-p36.13, which is well known to show highly significant linkage with the genesis and/or progression of human neuroblastoma. Southern blot analysis on tumor DNA from 26 patients with neuroblastoma has detected three patients showing genomic rearrangement or deletion within or closely linked to the DAN gene locus. Collectively, we propose that human DAN gene is a possible candidate for a tumor suppressor gene of human neuroblastoma.

Angiotensin-converting enzyme inhibitor increases angiotensin type 1A receptor gene expression in aortic smooth muscle cells of spontaneously hypertensive rats.

To examine the regulation of angiotensin receptors in vascular smooth muscle cells, we studied the effects of antihypertensive drugs on angiotensin type 1A (AT1A) receptor gene expression in aortic smooth muscle cells (ASMCs) from spontaneously hypertensive rats (SHRs) using both ribonuclease protection assay and reverse-transcription polymerase chain reaction. An increase in AT1A receptor gene expression in ASMCs of SHRs was induced by treatment with an angiotensin converting enzyme inhibitor (enalapril) for 2 weeks and 4 weeks, but not by other types of antihypertensive drugs such as alpha-blocker (doxazosin), alpha, beta-blocker (arotinolol), Ca antagonist (nicardipine) or vascular smooth muscle relaxant (hydralazine). Since all antihypertensive drugs lowered the blood pressure of the rats almost equally, our results suggest that AT1A receptor gene expression in ASMCs of SHRs may be regulated by the vascular renin-angiotensin system.

Cardiac output and peripheral resistance in strains of rats sensitive and resistant to NaCl hypertension.

The interrelationship of blood pressure, cardiac output, and peripheral resistance was studied in Dahl "S" and "R" rats after 3 days on a high (8%) NaCl diet. Both "S" and "R" rats were normotensive when fed a normal (0.3%) NaCl diet. After 3 days of the high NaCl diet, the "R" rats remained normotensive (BP 112 mm Hg), while the "S" rats had an elevation of arterial pressure (BP 133 mm Hg) (p less than 0.001). The cardiac outputs of both "S" and "R" rats were similar on the low NaCl diet. After 3 days of high NaCl feeding, the cardiac output of the "R" rats rose 18% above the "R" control level (p less than 0.0001), while the peripheral resistance declined 14% below the "R" control level (p less than 0.005), and the blood pressure (BP) did not change, a pattern quite contrary to the concept of "whole-body" autoregulation. With a similar 3-day high NaCl feeding in "S" rats, cardiac output (p less than 0.005) and peripheral resistance (p less than 0.05) both increased 10%, while BP rose 20%. After 7 days of high NaCl feeding, the cardiac output of the "S" rats had returned to normal, while blood pressure and peripheral resistance both continued to be elevated. This pattern of response in "S" rats could be compatible with the concept of "whole-body" autoregulation. However, since both NaCl hypertension and Goldblatt hypertension can occur in settings in which "whole-body" autoregulation appears not be to causally related, one cannot be certain whether "whole-body" autoregulation is playing a causal role in the mechanism of NaCl-induced hypertension in "S" rats. It is a striking dichotomy that 3 days of high salt feeding produces vasoconstriction in "S" rats and vasodilation in "R" rats.

Platelet thromboxane inhibition by plasma polypeptides in prehypertensive Dahl rats.

Plasma from Dahl rats susceptible to salt-induced hypertension (Dahl S rats) contains inhibitory factors that reduce the release of thromboxane A2 from thrombin-activated platelets. Platelet-rich plasma from Dahl S rats on either low salt (0.11 or 0.3% NaCl) or high salt (4% NaCl) diets released about 50% less thromboxane A2 than comparable plasma from Dahl rats that are resistant to hypertension (Dahl R rats). This inhibitory activity was present even in the blood of 4-week-old completely normotensive Dahl S rats on a diet containing 0.11% low NaCl. The inhibitory activity could be transmitted to platelets of normal Sprague-Dawley rats by incubating these platelets in boiled and dialyzed plasma from Dahl S rats. Moreover, the inhibitory activity could be completely washed off the Dahl S platelets by incubation in Dahl R plasma. Thus, Dahl S plasma contains inhibitory factors that reduce platelet thromboxane A2 release. The factors are found in low concentrations even in Dahl R plasma; and in Dahl S or Dahl R plasma the factors are increased 25 to 32% by a 4% high NaCl diet. Digestion of Dahl S and Dahl R plasma with either trypsin or chymotrypsin destroyed the inhibitory factors, which have a molecular weight between 2,000 and 3,500. Twenty-four hours after bilateral nephrectomy, dialyzed plasma from both Dahl S and Dahl R rats was completely devoid of thromboxane A2 inhibitory activity. Thus, the factors appear to be heat-stable polypeptides either produced in the kidney or greatly influenced by the presence of renal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium feeding reduces hyperactive central nervous system pressor responses in Dahl salt-sensitive rats.

Dahl showed that feeding KCl prevents the rise in blood pressure caused by a high NaCl diet in salt-sensitive Dahl "S" rats. Such S rats when normotensive on a low NaCl diet have a 2 to 3 times greater pressor response than Dahl "R" rats to intracerebroventricular hypertonic saline (600 mOsm/liter) or angiotensin II (AII) (500 ng). Does dietary KCl prevent NaCl hypertension in S rats partly by abolishing these hyperactive central nervous system (CNS) pressor responses? The effect of potassium-loading on CNS pressor responses was studied in S rats on a low (0.3%) NaCl diet. Drinking a 2% KCl solution reduced the CNS pressor responses in S rats to both AII and hypertonic saline by 44% (p less than 0.025) and brought them down almost as low as in R rats. KCl added to the low NaCl dry diet also decreased the CNS pressor responses in S rats to AII and to hypertonic saline by 39% (p less than 0.01) and 59% (p less than 0.02) respectively. K-citrate added to the low NaCl diet was generally as effective as KCl in reducing CNS pressor responses. K-citrate reduced the angiotensin pressor response by 44% (p less than 0.001) and the hypertonic saline pressor response by 46% (p less than 0.05). Thus, potassium feeding greatly diminished the hyperactive CNS pressor responses in S rats. This CNS action may well explain a good part of the protective effect of KCl against NaCl hypertension in S rats.

Dahl S rats have increased natriuretic factor in atria but are markedly hyporesponsive to it.

Three experiments were carried out to determine whether atrial natriuretic factor (ANF) plays a part in Dahl hypertension. Results showed that ANF from both atria from 13 Dahl salt-sensitive (S) rats that had been fed a 4% NaCl diet for 12 weeks induced an average peak Na excretion of 23.0 muEq/min/g kidney in 13 Sprague-Dawley (SD) recipients vs 12.6 from atria from 13 salt-resistant (R) rats fed 4% NaCl (-45%, p less than 0.01), possibly indicating greater ANF secretion in S rats in order to enhance a reduced Na excretion. In 13 R rats, the ANF content in both atria increased from 14.0 after a 0.11% NaCl diet to 23.7 after 5 days of 4% NaCl diet (p less than 0.001) and then back to 12.6 after 12 weeks of 4% NaCl (p less than 0.001). Thus, ANF almost doubled after brief Na loading but returned to normal during continued Na loading. In S rats with a tendency to Na retention, ANF was elevated to about 23 in all three periods. ANF produced a 130-fold increase in natriuresis and a renal papillary plasma flow ( RPPF ) of 30.8 ml/min/100 g, 41% above the control level of 21.7, p less than 0.001. The marked increase in RPPF is very likely a partial cause of the natriuresis. A constant amount of ANF was continuously infused intravenously into 10 S rats and nine R rats all on 0.11% NaCl diets. Mean Na excretions in R and S were 5.3 and 4.6 muEq/min/100 g kidney before ANF.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertension-producing factor in serum of hypertensive Dahl salt-sensitive rats.

To investigate whether serum in hypertensive Dahl salt-sensitive rats (S rats) contains a hypertensinogenic substance, we examined the effects of repeated injections of serum from such S rats on blood pressure (BP) and pressor responses. Serum was collected from either hypertensive or normotensive S rats (fed an 8% or 0.11% NaCl diet, respectively) and injected into uninephrectomized recipient S rats for 2 weeks (0.45 ml, twice a day, i.v.). Serum from hypertensive rats injected for 14 days significantly increased BP by 14 mm Hg (143 vs 129, p less than 0.05), pressor responses to angiotensin II (ANGII) by 45% (p less than 0.005), pressor responses to norepinephrine (NE) by 38% (p less than 0.025), and Na concentration in the aortic wall of recipient rats by 5.9% (p less than 0.05), compared to the effects of the injection of serum from normotensive S rats. These results imply that hypertensive S serum contains a hypertensinogenic substance and that this serum factor produces a mild hypertension in the recipient rats and also contributes importantly to the hypertension in donor S rats. Dahl salt-resistant rats (R rats) on either 8% or 0.11% NaCl had normal BP. Their sera produced no differences in BP or in pressor responses in recipient rats. Hence 8% NaCl, which produced no hypertension, also induced no hypertensinogenic serum factors in R rats. We sought to determine whether nephrectomy would alter these humoral factors. The BP averaged 139 mm Hg in rats receiving normotensive sham-nephrectomized S serum vs 154 in those receiving hypertensive sham-nephrectomized S serum, 15 mm Hg higher (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium reduces cerebral hemorrhage and death rate in hypertensive rats, even when blood pressure is not lowered.

In a study of the effects of K+ in stroke prone spontaneously hypertensive rats, adding K+ to normal chow was found to reduce the mortality from 83% to 2%, a 98% reduction. An 86% reduction in mortality occurred even when blood pressure was virtually equal in the two stroke prone spontaneously hypertensive groups being compared. Dietary K+ supplements also reduced mortality in hypertensive Dahl salt-sensitive rats from 55% to 4%, a 93% reduction. There was an 87% reduction in mortality even when blood pressure was equal in the Dahl salt-sensitive groups being compared. The added dietary K+ decreased blood pressure moderately in stroke prone spontaneously hypertensive rats and modestly in Dahl salt-sensitive rats, which probably contributed to the reduced death rate. More importantly, however, the added K+ seemed to prevent severe lesions in cerebral arteries and deaths even when blood pressure lowering was eliminated as a protective factor. In another group of stroke prone spontaneously hypertensive rats, there was a 40% incidence of cerebral hemorrhage in surviving rats not receiving K+ supplements and no incidence of cerebral hemorrhage in similar surviving rats receiving K+ supplements, which suggests that K+ supplements confer protection against brain hemorrhage.

Effect of salt on prostaglandin metabolism in hypertension-prone and -resistant Dahl rats.

The effect of high salt intake on vascular and renomedullary prostaglandin (PG) synthesis was compared in Sprague-Dawley and salt-sensitive (S) and -resistant (R) Dahl rats. Animals were given a diet containing either 0.6% or 8% NaCl starting at 5 weeks of age, and were sacrificed 6 weeks later. Systolic blood pressure of S rats increased to 220 +/- 7 mm Hg but was unaffected in R and Sprague-Dawley rats. Prostaglandin synthesis was studied in aortic rings and renomedullary microsomes using 14C-arachidonate as substrate. [3H]PGE2 degradation was measured in the renocortical cytosol. In Sprague-Dawley and R rats, aortic PGI2 synthesis was not affected by high salt intake, while a significant increase compared to animals on 0.6% NaCl (from 608 +/- 84 to 992 +/-108 pmoles/60 min, p less than 0.05) was noted in S rats. Enhancement of PGI2 synthesis in S rats may be secondary to the hypertension. Salt-loading consistently stimulated renomedullary PGE2 synthesis in all three animal groups. S rats, however, had the lowest PG synthesis in renal medullas compared to Sprague-Dawley and R rats when placed on either diet. Thus, even after 6 weeks on high salt, S rats did not reach the levels of PGE2 synthesis seen in R or Sprague-Dawley rats on regular diet. The activity of cortical 15-hydroxyprostaglandin dehydrogenase was increased by salt-loading in S and Sprague-Dawley, but not in R rats. R rats had lower dehydrogenase activity than the other two groups when placed on either diet. The observed differences in PG synthesis and catabolism will tend to maintain the net output of renal PGs highest in R and lowest in S rats. These differences correlate with the reported differences in renal papillary flow between these two rat strains and may be relevant to their susceptibility or resistance to hypertension in response to salt.

Cardiac hypertrophy and performance of Dahl hypertensive rats on graded salt diets.

The relationship between arterial pressure and left ventricular (LV) functional capacity and LV mass during the natural development of cardiac hypertrophy was assessed in Dahl-resistant (R) and -sensitive (S) hypertensive rats maintained on three dietary NaCl regimens (0.4%, 4.0%, and 8.0% for 9 weeks, then 4.0%) from 5 until 20 weeks of age. In R rats, arterial pressure and LV mass were unaffected by diet. In contrast, S rats demonstrated levels of arterial pressure and LV hypertrophy that were graded according to dietary NaCl. Hemodynamic studies on rats under ether anesthesia demonstrated that the graded pressure elevation in S rats was produced by corresponding increases in total peripheral resistance, as cardiac output did not vary. During acute volume loading, the S rats on all diets achieved the same maximum stroke volume as did R rats, despite the marked increase in the arterial pressure of S rats. An analysis of the ejection fraction/afterload relationship demonstrated preserved contractile state. The ability of the left ventricle to generate pressure was increased in S rats in direct proportion to the degree of LV hypertrophy. Thus, in young adult S rats, cardiac performance was well compensated since pump and contractile functions were maintained and pressure-generating capacity was increased in relation to the degree of LV hypertrophy.

How is the NaCl signal transmitted in NaCl-induced hypertension?

Is the NaCl signal perceived as a small increase in the concentration of NaCl in extracellular fluid? We used 8 g NaCl/100 g soluble nutrients and fed only a hypertonic (1.4% NaCl) or a hypotonic (0.45% NaCl) drink to Dahl salt-sensitive (DS) rats. After 12 weeks, 11 rats receiving the hypertonic drink had a mean blood pressure of 195 mm Hg versus 195 mm Hg in 12 rats receiving the hypotonic drink. Thus, the high-NaCl signal seems unrelated to a higher NaCl concentration in extracellular fluid, thereby suggesting volume signals. Most volume controls are near the third brain ventricle (3V). As a working hypothesis, high dietary NaCl may swell the tissues surrounding 3V, which is slitlike. Such swelling would partially close the upper part of the slit and cause ependymal cells and nerve fibers on opposite walls to touch, possibly leading to hypertension in susceptible humans or rats. To test this, we stereotaxically blocked the aqueduct with inert silicone to produce hydrocephalus of 3V in DS rats and thus prevent ependymal cells and nerve fibers from touching. After blocking or sham-blocking the aqueduct, either a 6% NaCl diet or a 0.23% NaCl diet was started. Intra-arterial blood pressure was taken after 6 weeks. A group of 28 sham-blocked rats and a group of 29 blocked rats, all fed a 0.23% low NaCl diet, had equal blood pressures averaging 130 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)