Intracerebroventricular infusion of aldosterone induces hypertension in rats.

There is suggestive evidence for the direct participation of mineralocorticoids in the production of centrally mediated hypertension. Unilaterally nephrectomized Sprague-Dawley rats received a continuous infusion for 30 days using implanted osmotic minipumps with 1) artificial spinal fluid (CSF) intracerebroventricularly (icvt); 2) 0.005 micrograms aldosterone/h icvt; 3) 0.005 micrograms aldosterone/h sc; 4) 0.5 micrograms aldosterone/h sc. There was no significant difference between the groups in average weight gain (52 +/- 2 g) or organ weight to body weight, nor was urine volume increased above normal except in the group receiving the high sc dose of aldosterone. Blood pressure was significantly elevated only in those animals receiving 0.005 micrograms aldosterone/h icvt and 0.5 micrograms aldosterone/h sc. A second experiment was done using a specific spironolactone-type mineralocorticoid antagonist, prorenone. The rats were grouped as follows: 1) CSF icvt; 2) 0.005 micrograms/h aldosterone icvt; 3) 0.005 micrograms/h aldosterone plus 0.005 micrograms/h prorenone icvt; 4) 0.005 micrograms/h aldosterone plus 0.02 micrograms/h prorenone icvt; 5) 0.02 micrograms/h prorenone icvt. This study confirmed that this minute dose of aldosterone infused icvt produced a statistically significant increase in blood pressure with no increase in urine volume. Both the low, 0.005 micrograms/h, and high, 0.02 micrograms/h, doses of prorenone antagonized the pressor effect of aldosterone when infused with aldosterone into the lateral cerebral ventricle. The groups receiving 0.02 micrograms/h prorenone icvt or CSF icvt did not differ significantly in those parameters measured. A dose of aldosterone that was too small to produce changes in blood pressure when infused systemically was found to produce hypertension without polydypsia/polyuria when infused intrathecally. This pressor effect could be blocked by the concomitant infusion of a specific antagonist, prorenone. The study presented offers strong circumstantial evidence supporting a direct hypertensinogenic effect of aldosterone within the central nervous system.

A reevaluation of the mineralocorticoid and hypertensinogenic potential of 19-hydroxyandrostenedione.

Experiments were done to evaluate whether 19-hydroxyandrostenedione (19-OH-A) is a steroid which produces hypertension and amplifies the mineralocorticoid effects of aldosterone. No intrinsic mineralocorticoid or adosterone-amplifying effect was found in several bioassays using adrenalectomized rats at doses of 100 micrograms 19-OH-A and/or 0.1 microgram aldosterone or in assays in which urine electrolytes were measured daily during a 14-day continuous infusion of 100 micrograms/day 19-OH-A. In two different experiments, the twice weekly administration of 0.1-1 mg 19-OH-A in oil to intact rats' drinking water failed to produce a change in blood pressure after 6 weeks. When one kidney was removed from these rats and 0.9% saline was substituted for drinking water, the blood pressures of rats receiving 1 mg twice weekly of 19-OH-A became significantly elevated in only one of the two experiments. At the end of 10 weeks, the average 19-OH-A pressure (139 mmHg) was significantly (P less than 0.01) greater than average control pressure (114 mmHg), but significantly (P less than 0.01) less than the mean average pressure of rats receiving 5 mg deoxycorticosterone acetate (DOCA) (161 mmHg). In another experiment uninephrectomized rats received 0.5 mg 19-OH-A or 1 mg DOCA 3 times a week. After 5 weeks the pressure of those receiving 19-OH-A was 149 mmHg, significantly greater (P less than 0.01) than controls (123 mmHg), but significantly less (P less than 0.01) than that of those rats receiving DOCA (189 mmHg). Our data failed to confirm the aldosterone amplifying effect of 19-OH-A. We did obtain a small elevation of arterial pressure in unilaterally nephrectomized rats receiving saline to drink. The differences between our results and those reported for this compound are not readily apparent, but may be related to genetic differences within the Sprague-Dawley strain used in both studies and/or to diet.

RU-26988--a new tool for the study of the mineralocorticoid receptor.

Aldosterone binds to two renal cytosol receptors, one with high affinity and low capacity (Type I or mineralocorticoid) and another with lower affinity but greater capacity (Type II or glucocorticoid receptor). One of the ways to study Type I receptors isolated from Type II receptors is to block the latter with a steroid which shows high affinity for the glucocorticoid receptor and low affinity for the mineralocorticoid receptor. Dexamethasone has been used widely for this purpose but previous investigations and this study have found that dexamethasone competes with [3H]aldosterone for Type I receptor binding with a relative activity of 26% that of aldosterone and therefore is less than ideal as a glucocorticoid receptor blocker. RU-26988 (11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6-trien-20-yn-21-methyl-3-one) is a recently synthesized glucocorticoid that shows very low affinity for the mineralocorticoid receptor. RU-26988 competes with [3H]aldosterone and [3H]2 alpha-methyl-9 alpha-fluorocortisol, a powerful mineralocorticoid, for the cytosol receptor from renal slices of adrenalectomized rats with a relative potency of less than 0.5% in comparison to unlabeled aldosterone and 2 alpha-methyl-9 alpha-fluorocortisol. RU-26988 has over twice the ability of unlabeled dexamethasone to compete with [3H]dexamethasone for binding to the renal cytosol glucocorticoid receptor. Scatchard analysis of [3H]aldosterone binding to rat renal cytosol showed two receptors, one with a calculated dissociation constant (Kd) of 2.81 X 10(-9) M and a second with a calculated Kd of 3.33 X 10(-8) M. The addition of a 100-fold concentration of RU-26988 produced a single line with a Kd of 5.02 X 10(-10) M indicating that the specific blocking of the glucocorticoid receptors allows an accurate determination of the kinetic parameters of the mineralocorticoid receptor by itself. Scatchard analysis of [3H]2 alpha-methyl-9 alpha-fluorocortisol binding produced a straight line with a Kd of 3.7 X 10(-9) M, such as would be produced if it were binding to only one single class of receptors. However, when an excess of RU-26988 was added to block the glucocorticoid receptor, a different straight line was produced by Scatchard's analysis with a Kd of 3.78 X 10(-10) M. Whereas the explanation for this is not apparent, it may be that the much larger concentration of glucocorticoid receptors, for which 2 alpha-methyl-9 alpha-fluorocortisol also has a very great affinity, masks the binding to the mineralocorticoid receptors.

19-Nordeoxycorticosterone excretion in male and female inbred salt-sensitive (S/JR) and salt-resistant (R/JR) Dahl rats.

Rats were selectively bred for susceptibility (S) and resistance (R) to the hypertensinogenic effects of excess salt intake by Dahl and further inbred to virtual homozygosity by Rapp (S/JR and R/JR). The S strain has been shown to have a mutation of the cytochrome P-450-dependent 11 beta,18-hydroxylase resulting in the enhanced production of 18-hydroxydeoxycorticosterone (18-OH-DOC) compared to that of the R strain. It is known that this enzyme is also responsible for the hydroxylation of deoxycorticosterone at the 19 position to produce 19-hydroxydeoxycorticosterone. Recently, the excretion of 19-nordeoxycorticosterone (19-nor-DOC), a potent mineralocorticoid, has been shown to be markedly increased in S/JR females compared to that in R/JR females consuming a high sodium diet. While the S/JR rat is spontaneously hypertensive, the course of the disease is greatly accelerated and exacerbated by a high sodium diet. If, indeed, 19-nor-DOC is responsible for the spontaneous hypertension in the S/JR rat, then its production should also be higher in the S/JR rat consuming a normal salt diet. Furthermore, since its production is suppressed by NaCl intake, the excretion should be even higher when not suppressed by a high sodium diet. We measured the urinary excretion of 19-nor-DOC, 18-OH-DOC, and corticosterone in male and female S/JR and R/JR rats consuming a normal sodium diet. The excretions of corticosterone and 18-OH-DOC were significantly higher by S/JR of both sexes than by R/JR, with the excretion by female rats being higher than that by male rats within the same strain. The hierarchy of excretion rates of 19-nor-DOC was: S/JR females greater than R/JR females greater than S/JR males greater than R/JR male rats. These studies indicate that while S/JR rats of both sexes develop higher blood pressures than the R/JR even on a standard salt intake, the excretion of 19-nor-DOC does not correlate well with their blood pressure elevation, since the normotensive female R/JR rat excretes significantly higher quantities of 19-nor-DOC than the hypertensive male S/JR rat. Thus, it is unclear whether 19-nor-DOC is playing a significant role in the pathogenesis of the hypertension in the S/JR rat. It also remains unknown whether the renal site of formation of 19-nor-DOC allows access to the mineralocorticoid target sites in the kidney.

Radioimmunoassay for urinary free 19-nor-deoxycorticosterone: effects of adrenocorticotropin and sodium depletion.

A RIA for urinary free deoxycorticosterone (DOC) and 19-nor-deoxycorticosterone [21-hydroxy-19-nor-4-pregnene-3,20-dione (19-nor-DOC)] was developed and used to measure urinary free DOC and 19-nor-DOC in rats kept on normal and low sodium diets and before and after ACTH administration. The RIA required a preliminary high performance liquid chromatography purification using a Lichrosorb diol column before the eluates were assayed using highly specific antibodies. The intraassay variability was 9.8% for DOC and 10% for 19-nor-DOC; the interassay variabilities were 19% and 16%, respectively. Urinary free DOC excretion was 0.71 +/- 0.15 ng/day (mean +/- SD) in rats maintained on a normal sodium diet and 1.42 +/- 0.46 ng/day after 3 days of a sodium-restricted diet. Urinary free 19-nor-DOC was 10.5 +/- 5.5 ng/day in rats kept on the normal sodium diet and 28.6 +/- 11.5 ng/dl after 3 days of a sodium-restricted diet. ACTH increased the excretion of urinary free DOC from 0.72 +/- 0.2 to 4.62 +/- 2.3 ng/day and that of 19-nor-DOC from 5.37 +/- 2.8 to 10.15 +/- 2.5 ng/day. These data indicate that the adrenal precursor of 19-nor-DOC probably comes from both the zona fasciculata and the glomerulosa to explain the ACTH and sodium depletion responses.

Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11 beta-hydroxysteroid dehydrogenase.

A pcDNAI adult rat kidney complementary DNA (cDNA) library was screened using a sheep 11-hydroxysteroid dehydrogenase 2 (11 beta HSD-2) probe, and the isolated clones were sequenced. The 5'-end of the cDNA was determined by 5'-rapid amplification of cDNA ends. The rat 11 beta HSD-2 cDNA is 1864 base pair (bp) long. It consists of a 5'-untranslated region of 126 bp, an open reading frame of 1203 bp, and a 3'-untranslated region of 535 bp. The predicted protein contains 400 amino acid residues, with a calculated mol wt of 43,700. The rat 11 beta HSD-2 protein sequence is 85% homologous to human 11 beta HSD-2 and 76% to sheep 11 beta HSD-2. Expression of 11 beta HSD-2 messenger RNA by Northern blot and reverse transcription-polymerase chain reaction was high in kidney, distal colon, and adrenal and lower in the lung, hypothalamus, hippocampus, and midbrain. The rat 11 beta HSD-2 was transiently transfected into modified Chinese hamster ovary cells. Cells transfected with the 11 beta HSD-2 cDNA converted corticosterone into 11-dehydrocorticosterone. Conversion of corticosterone to 11-dehydrocorticosterone was NAD+ dependent and had a Km of 10.1 +/- 2.1 nM. In conclusion, we have cloned a rat NAD(+)-dependent 11 beta HSD with tissue distribution and kinetic characteristics suggesting that it could play a significant role in mineralocorticoid receptor selectivity.

Mineralocorticoid and hypertensive effects of 19-nor-progesterone.

The mineralocorticoid potency of 19-nor-progesterone was evaluated by both its effect on electrolyte excretion in adrenalectomized animals and its ability to cause hypertension and electrolyte changes in mononephrectomized, salt-loaded rats. The mineralocorticoid activity, measured using an adrenalectomized rat bioassay, indicated that 19-nor-progesterone was 2.5% as potent as aldosterone but did not antagonize the effect of aldosterone when both were administered. In mononephrectomized rats, the daily administration of 1 mg/day quickly caused an enhanced consumption of 1% saline and induced severe hypertension within 3-4 weeks. Some severely hypertensive animals had marked anemia, but other did not; as a group they were found to have hypernatremia and hypokalemia. Hypertensive animals were found during life to display a relative hypothermia and, at necropsy, to have heart and kidney enlargement with severe and extensive vascular lesions in both organs, but not adrenal hypertrophy. It is concluded that 19-nor-progesterone has the characteristics of a potent mineralocorticoid and, as such, is capable of causing hypertension. It is not yet clear why this should be accompanied by hypothermia.

Identification of 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone as products of deoxycorticosterone metabolism by rat adrenals.

The formation of 19-hydroxydeoxycorticosterone (19,21-dihydroxy-4-pregnen-3,20-dione), 19-oxo-deoxycorticosterone (21-hydroxy-4-pregnen-3,19,20-trione), and 19-oic-deoxycorticosterone (19-oic-21-hydroxy-4-pregnen-3,20-dione) from precursor deoxycorticosterone by adrenal glands obtained from intact rats and from rats undergoing adrenal regeneration was demonstrated. These metabolites were isopolar with corresponding authentic steroid standards on thin layer chromatography, gas chromatography, and high pressure liquid chromatography. They were further characterized by either mass spectrometry or gas chromatography-mass spectrometry. Therefore, rat adrenals have the enzymes required to convert deoxycorticosterone to 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone; however, rat adrenals do not convert deoxycorticosterone or any of the oxygenated metabolites to 19-nor-deoxycorticosterone (21-hydroxy-19-nor-4-pregnen-3,20-dione). It is possible, however, that 19-nor-deoxycorticosterone is formed at peripheral sites from the oxygenated deoxycorticosterone precursors.

Receptor binding and biological activity of 18-hydroxycortisol.

Recently, 18-hydroxycortisol (11 beta,17 alpha,18,21-tetrahydroxy-4-pregnene-3,20-dione) was isolated and identified from extracts of urine and adrenal incubates of patients with primary aldosteronism. The receptor-binding activity to the renal gluco- and mineralocorticoid receptors and its biological activity as a glucocorticoid and mineralocorticoid were investigated using synthetic 18-hydroxycortisol. The ability of 18-hydroxycortisol to compete with [3H]aldosterone for renal binding to the receptor was 0.13% that of unlabeled aldosterone. The addition of a specific glucocorticoid, RU-26988 (11 beta,17-dihydroxy-21-methyl-17 alpha-pregna-1,4,6-triene-20-yn-3-one) decreased the competing ability to 0.02%, indicating significant binding to the glucocorticoid receptor. The ability to compete with [3H]dexamethasone for the renal cytoplasmic glucocorticoid receptor was 0.1% that of unlabeled dexamethasone. The mineralocorticoid activity of 18-hydroxycortisol was undetectable. Its glucocorticoid activity using an in vitro bioassay based on the induction of tyrosine aminotransferase in the HTC cell was detectable at 10(-5) M, but was too low for adequate quantification. In a second in vitro glucocorticoid bioassay, inhibition of cell growth of the L929 fibroblast, 18-hydroxycortisol also showed minimal activity. In summary, it is unlikely that 18-hydroxycortisol plays a role in the metabolic syndrome in those patients who produce it in excess due to its inactivity as a gluco- or mineralocorticoid.

Renal receptor-binding activity of reduced metabolites of aldosterone: evidence for a mineralocorticoid effect outside of the classic aldosterone receptor system.

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.

11 beta-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: specific inhibition by 11 alpha-hydroxyprogesterone.

The 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11 beta HSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11 beta HSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 +/- 3.1 nM, and that for NAD+ was approximately 8 microM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity. 11 alpha-Hydroxyprogesterone (11 alpha OH-P) was an order of magnitude more potent a competitive inhibitor of the 11 beta HSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 = 0.9 vs. 15 nM). 11 beta OH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5 alpha-pregnandione and 5 beta-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11 alpha OH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11 alpha OH-P was not metabolized by 11 beta HSD-2. We were unable to demonstrate the presence of 11 alpha OH-P in human urine. In conclusion, a cell line stably transfected with the rat 11 beta HSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11 alpha OH-P was found to be a potent relatively specific inhibitor of the 11 beta HSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11 beta HSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme.

Receptor binding and biological activity of 18-oxocortisol.

It has been recently demonstrated that cortisol can be metabolized, producing 18-hydroxycortisol and 18-oxocortisol, following the same pathway by which corticosterone is transformed into 18-hydroxycorticosterone and aldosterone. The influence of a hydroxy group in the 17 alpha position of aldosterone or an aldehyde (actually 11-18 hemiacetal) in the 13-methyl of cortisol on the mineralocorticoid and glucocorticoid activities were studied and compared with the parent steroids. The ability of 18-oxocortisol to complete with [3H]aldosterone for binding to the cytosol receptor of rat renal slices was 8.1% in comparison to unlabeled aldosterone. The addition of a specific glucocorticoid 11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6- trien-20-yn-21-methyl-3-one decreased this binding to 5.6%. The ability of 18-oxocortisol to compete with [3H]dexamethasone for binding to the renal cytosol receptor was 0.2% that of unlabeled dexamethasone and in the HTC whole cell assay was 1.06% and 3.8% that of unlabeled dexamethasone and cortisol, respectively. The mineralocorticoid activity of 18-oxocortisol in the adrenalectomized rat bioassay was 0.6% that of aldosterone. The glucocorticoid activity in in vitro bioassays was 3.1% compared with that of a cortisol when the induction of tyrosine aminotransferase in HTC cells was measured and 4% when the inhibition of fibroblast L-929 growth was measured. The significance of 18-oxocortisol in the pathogenesis of the hypertension in patients with primary aldosteronism is still unclear.

Aldosterone biosynthesis in the rat brain.

Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain minces with [1,2(3)H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 microM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.

Maternal hypertension and progeny blood pressure: role of aldosterone and 11beta-HSD.

Epidemiological and experimental evidence suggests that gestational events modulate the level of blood pressure that will be "normal" for the individual as an adult. Glucocorticoid excess during gestation is associated with low birth weight, a large placenta, and adult hypertension in humans and animals. It has been proposed that the deficiency in placental 11beta-hydroxysteroid dehydrogenase activity in humans produces a gestational hormonal milieu, notwithstanding normal circulating levels of glucocorticoids, that predisposes the adult progeny to hypertension. Animal studies indicate that maternal hypertension, excess glucocorticoids, and hydroxysteroid dehydrogenase inhibition program adult blood pressure. Blood pressures of Sprague-Dawley rat dams were manipulated during gestation with continuous intracerebroventricular infusions of vehicle, aldosterone, 11alpha-hydroxyprogesterone, or carbenoxolone at doses known to produce hypertension with no renal effects or with subcutaneous infusions of larger, equally hypertensinogenic doses that produce systemic effects. Blood pressures of all treated dams were significantly greater (P<0.01) during gestation than those of the vehicle ICV control rats but not significantly different from each other. The blood pressures of both male and female progeny (n>/=6 per group, comprising representatives from at least 4 litters) were measured after 6 weeks of age. No significant difference was found in the blood pressure of the pups regardless of the maternal gestational blood pressure or treatment with an enzyme inhibitor, even after high-salt diet challenge.

Modulation of blood pressure in the Dahl SS/jr rat by embryo transfer.

Gestational hypertension and malnutrition are associated with hypertension and ischemic heart disease in the adult human. The impact of the gestational environment on the adult blood pressure in two well-characterized genetically homogeneous rat strains, the hypertensive SS/jr and normotensive SR/jr, was studied by cross-fostering within 6 hours of birth and by embryo transplantation with the recipient dam nursing the transplanted pups. Systolic blood pressure (BP) was measured by tail-cuff plethysmography twice a week after the age of 7 weeks. The lactational environment (cross-fostering) had no effect on blood pressure. Embryo transfer between like strains had no effect on the development of hypertension, nor did the BP of R transferred to S (RetS) differ from that of normal R or RetR. At 7 weeks of age, the BP of SetR was significantly lower than that of S or SetS (P<.01) and was similar to that of RetR and R. With age, the blood pressures of the S, SetS and SetR increased at approximately the same rate but from a significantly different baseline. Salt-sensitivity in the S and resistance in the R were not altered. The protective effect of the R gestational environment on SetR female BP was abrogated during whelping and lactation. Embryo transfer and cross-fostering did not alter the weight of rats older than 7 weeks. Because the BP of the R dams were significantly lower than that of the S dams, these studies do not distinguish between the effects of the R dams' lower blood pressure per se and hormonal influences of the R uterus on the S blood pressure phenotype.

Elevated urinary excretion of 18-oxocortisol in glucocorticoid-suppressible aldosteronism.

A radioimmunoassay procedure for the measurement of urinary 18-oxocortisol was developed. The antibody was raised against 18-oxocortisol 3-carboxymethyloxime-BSA and had relatively high specificity, except for aldosterone (26.3%). The RIA required a preliminary HPLC purification using a Lichrosorb diol column eluted with toluene:acetonitrile:isopropanol:acetic acid (83:11.9:5.1:0.01). The eluate portion corresponding to 18-oxocortisol was evaporated and subjected to RIA. The RIA procedure had an intraassay variability of 11% when using a pool containing 10.8 micrograms/24 hr (n = 6) and 17% with a pool containing 3.28 micrograms/24 hr. The interassay variability was 11% (n = 4). The recovery of added 18-oxocortisol was 90 +/- 10%. The urinary excretion of 18-oxocortisol in 22 white normal subjects was 3.26 +/- 1.98 (SD) micrograms/24 hr (range 0.8 to 7.1 micrograms/24 hr). The mean excretion of 18-oxocortisol in 4 patients with glucocorticoid-suppressible aldosteronism (GSA) was 38.6 micrograms/24 hr (range 25.5 to 54.6 micrograms/24 hr). The excretion of 18-oxocortisol in 3 patients with adenomas producing primary aldosteronism (APA) varied between 11.1 to 17.3 micrograms/24 hr and in 3 patients with idiopathic aldosteronism (IA) varied between 2.5 to 10.6 micrograms/24 hr. 18-Oxocortisol excretion is increased markedly in the urine of patients with GSA: what role this relatively weak mineralocorticoid plays in the pathogenesis of their hypertension is unknown. Its elevation is probably a reflection of a postulated lack of involution of the 18-methyloxidase in the inner layers of the adrenal.

Dose-response studies of intracerebroventricular infusion of aldosterone in sensitized and non-sensitized rats.

We have shown previously that the intracerebroventricular (icvt) infusion of 5 ng/h aldosterone (ALD) in the sensitized rat (one kidney removed, 1% NaCl plus 0.15% KCl solution to drink) produced hypertension similar in amplitude and time of onset to a 100-fold dose administered subcutaneously (s.c.), while a 5-ng/h subcutaneous infusion had no effect on blood pressure (BP). Dose-response studies on the icvt infusion of ALD were carried out in sensitized and non-sensitized (intact, with tap water to drink) male Sprague-Dawley rats (SDR). In both studies, a control group received the diluent, artificial cerebrospinal fluid (CSF), icvt. In sensitized rats, the pressures became significantly (P less than 0.05) elevated at day 7 in those receiving 15 ng/h icvt, day 11 in those receiving 5 ng/h icvt and 500 ng/h s.c. and day 18 in those receiving 1.5 ng/h icvt. The indirect systolic BPs at day 20 of infusion were 119 +/- 0.8 (s.e.) mmHg for controls, 182 +/- 5 for 15 ng/h icvt, 140 +/- 2 mmHg for 5 ng/h icvt, 131 +/- 1 mmHg for 1.5 ng/h icvt, 125 +/- 1 mmHg for 0.5 ng/h, and 159 +/- 5 mmHg for 500 ng/h s.c. Recovery (removal of pumps and return to water to drink) for 18 days resulted in the return of normal pressures in all groups except the 15 ng/h, icvt group in which pressures remained slightly, but significantly elevated at 127 +/- 3 mmHg. In non-sensitized rats, the pressures became significantly elevated in animals receiving 45 ng/h icvt and 1 microgram/h s.c. by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereditary cataracts in the John Rapp inbred strain of Dahl salt-sensitive rat.

Cataracts were apparently fixed in the genome of a highly inbred strain of Dahl salt-sensitive (S/JR) rat during the course of the selection for hypertension. Cataracts were present in S/JR rats inbred for more than 40 generations, but were not present in any of the salt-resistant rats (R/JR) inbred and observed during the same time. Light and electron microscopic evaluation of 11 pairs of S/JR rat lenses revealed a large posterior capsular defect and marked degenerative changes in lens fibers in each case. While the reason for the posterior capsular break is unclear, the cataract is probably a manifestation of an abnormal recessive gene or a recent autosomal dominant mutation.

An alternatively spliced rat mineralocorticoid receptor mRNA causing truncation of the steroid binding domain.

We attempted to clone the putative 11-dehydrocorticosterone receptor by RT-PCR with two degenerate primers from highly homologous regions of the DNA and steroid binding domains of the receptor subfamily. In doing so, we have identified an alternatively spliced variant mRNA of the rat mineralocorticoid (MR) with a ten bp deletion in the C-terminal steroid binding domain. This deletion results in a truncated MR receptor of 807 amino acids in comparison to the wild type of 981 amino acids. The deletion variant was expressed in colon, kidney, heart, liver, aorta and brain tissues. The relative abundance of the deletion variant compared to the wild type MR was estimated to be 6% in rat kidney and 4% in hippocampus. This deletion was also detected in human kidney by RT-PCR. Site-directed mutagenesis was used to create the eukaryotic expression plasmid pCR3-rMRdel10 from the wild type for a transactivation assay using the luciferase reporter system in CV-1 cells. The deletion variant had the same baseline transactivation activity as the wild type MR, but did not respond to aldosterone or corticosterone stimulation. Co-transfection of MR with the deletion variant had no significant effect on transactivation activity of the MR, indicating that the deletion variant is unlikely to serve as a negative regulator of MR function.

Cloning and expression of the rat adrenal cytochrome P-450 11B3 (CYP11B3) enzyme cDNA: preferential 18-hydroxylation over 11 beta-hydroxylation of DOC.

The biosynthesis of glucocorticoids and mineralocorticoids in the rat adrenal cortex requires the action of two different cytochrome P450 11 beta-hydroxylases, CYP11B1 and CYP11B2, which are distributed in the zona fasciculata and glomerulosa, respectively. The existence of another cytochrome P450-11 beta gene, CYP11B3, was recently reported. Although CYP11B3 has similar gene structure and great homology to the CYP11B1 and -B2 genes, the CYP11B3 mRNA was not originally detected by reverse transcription-polymerase chain reaction (RT-PCR) and has only recently been cloned and detected from neonatal rat adrenals. Herein we demonstrate RT-PCR detection of CYP11B3 mRNA expressed in adult rat adrenal and brain tissues. The whole coding region of the CYP11B3 enzyme cDNA was cloned and sequenced. When transiently expressed in COS-7 cells the CYP11B3 converted deoxycorticosterone (DOC) to corticosterone and 18-hydroxydeoxycorticosterone, but not to 18-hydroxycorticosterone or aldosterone. It produced more 18-OH-DOC than corticosterone. A single mutation in CYP11B3 in which Gly-59 was replaced by Ser, reduced the enzymatic activity 5-6-fold. Furthermore, CYP11B3 mRNA expression is greater in neonatal, compared to adult rat adrenal glands.