Effects of water-deprivation on aldosterone production in Brattleboro male rats congenitally lacking vasopressin

Effects of water-deprivation on several metabolic parameters and on plasma aldosterone concentration have been investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI) and in male Long-Evans rats (LE) as controls. Two separate experiments were performed over a period of 72 hours: 1) to determine the global effect of water-deprivation, water deprived rats were compared with hydrated animals, 2) to elucidate the specific effect of dehydration alone, water-deprived rats were compared with similar food-restricted, but water-supplied DI and LE rats. In hydrated animals, plasma aldosterone concentration was close to 50% less in DI rats than in LE rats. After 72 hours, plasma aldosterone values increased mainly because of dehydration and this increase was greater in DI rats than in LE rats. At the same time, plasma aldosterone concentration remained lower in DI rats compared to LE rats. The changes in plasma aldosterone concentration after dehydration and possible reasons for the impairment of aldosterone production in DI rats are discussed.     

Effect of chronic vasopressin treatment on alcohol drinking of Brattleboro HZ and DI rats

Preference for alcohol was determined for three groups of male and female rats, 100–150 days old, comprised of: (1) Long Evans (LE); (2) LE-derived Brattleboro heterozygous (HZ); and (3) Brattleboro homozygous (DI) animals afflicted with diabetes insipidus due to vasopressin deficiency. Each alcohol drinking test was run over 11 days during which food, water and an ethyl alcohol solution, increased in concentration from 3% to 25%, were freely available. Following an initial preference screen, 100 milli-units of vasopressin tannate in oil was administered subcutaneously, during a second preference test, once per day to each animal. This treatment ameliorated the polydipsia-polyuria syndrome characteristic of the DI sub-strain of Brattleboro rat. Administration of the peptide to both the LE or HZ animals exerted no effect on g/kg intake nor on the proportional measure of alcohol to water. However, in the DI rat of either gender, vasopressin reduced the mean absolute gram intake of alcohol over concentrations to resemble that of the other LE and/or HZ groups. These results demonstrate that vasopressin serves to normalize the intake of alcohol in the DI rat by virtue of the elimination of the diabetic condition. However, since vasopressin fails to alter alcohol consumption of the HZ and LE rats, it would appear that this neuroactive peptide may play only a minor role in the CNS mechanisms governing the voluntary selection of alcohol.     

Production of corticosterone in male homozygous Brattleboro rats

Plasma concentration, metabolic clearance rate and in vitro adrenal production of corticosterone were measured in Brattleboro male rats homozygous for diabetes insipidus (DI) and in Long-Evans male rats (LE) as controls in resting conditions, under stress caused by pentobarbitone anesthesia and surgery and after three days water deprivation. In resting animals, plasma concentrations and in vitro adrenal production of corticosterone were higher in DI rats than in LE rats. Under pentobarbitone anesthesia and surgery, plasma concentrations and metabolic clearance rate of corticosterone were slightly but not statistically lower in DI rats; however, the in vivo production rate of corticosterone was significantly lower. After three days water deprivation, increasing plasma corticosterone level was consistently higher in DI than in Le rats. These results are not in favour of a reduced glucocorticoid activity of the adrenal of DI rats and of an important role played by vasopressin on the stimulation of the hypothalamopituitary adrenal activity at least in resting conditions; its role may depend upon stressful circumstances.     

Effects of synthetic atrial natriuretic factor (ANF) on renin-aldosterone axis in the conscious newborn calf

The effects of synthetic atrial natriuretic factor (ANF) on the renin-aldosterone axis were studied in fifteen 4-7 day-old male milk-fed calves divided into 3 groups of 5 animals each. Synthetic ANF intravenous (i.v.) administration (1.6 micrograms/kg body wt over 30 min) induced a transient significant fall in plasma renin activity (from 2.5 +/- 0.3 to 1.7 +/- 0.3 ng angiotensin l/ml/h; P less than 0.05) but failed to reduce basal plasma aldosterone levels in the first group of animals. Administration (i.v.) of angiotensin II (AII) (0.8 micrograms/kg body wt for 75 min) was accompanied by a progressive fall in plasma renin activity (from 2.2 +/- 0.3 to 0.8 +/- 0.1 ng angiotensin l/ml/h; P less than 0.01) and by an increase in plasma aldosterone levels (from 55 +/- 3 to 86 +/- 5 pg/ml; P less than 0.01) both in the second and the third groups; addition of ANF to AII infusion (AII: 0.5 mu/kg body wt for 45 min; AII: 0.3 micrograms/kg body wt and ANF 1.6 micrograms/kg body wt during 30 min) in the third group did not modify plasma renin activity or AII-stimulated plasma aldosterone levels when compared to the AII-treated group. These findings show that in the newborn calf ANF is able to reduce plasma renin activity but fails to affect basal and AII-stimulated plasma aldosterone levels, suggesting that the zona glomerulosa of the newborn adrenal cortex is insensitive to a diuretic, natriuretic and hypotensive dose of the atrial peptide.     

Secretion of aldosterone and corticosterone by the adrenals of Brattleboro rats in response to ACTH administration

The secretions of aldosterone and corticosterone in response to administration of 0,5 mUI of (1,24) ACTH (synacthène-Ciba) were measured in the adrenal venous blood of 15 Brattleboro female rats genetically lacking vasopressin and in 15 Long-Evans female rats, pretreated with dexamethasone. The secretions of aldosterone and corticosterone increased according to a similar profile in the two groups of animals: maximum values were 20-30 min. after ACTH injection; however the steroidogenic secretion of the adrenal cortex was always about 50% less in the Brattleboro female rats than in Long-Evans female rats. This result suggests mainly that vasopressin may be involved in the mechanisms which control the in vivo production of aldosterone by the adrenal glomerulosa cells.     

Angiotensin II increases ACTH release in the absence of endogenous arginine-vasopressin

Recent studies from our laboratory indicate a primary central site of action of Angiotensin II (AII) to release ACTH. The present studies were designed to test whether AII is able to release ACTH in vivo in a similar fashion in intact, cannulated, freely moving Long-Evans (LE) or in vasopressin (AVP)-deficient, Brattleboro (DI) female rats. The in vivo response to AII was compared with that elicited by synthetic CRF. AII injected i.v. (0.4 or 2 micrograms/100 g BW) induced a significant, dose-related increase in plasma ACTH values 5 and 15 min after injection, in both LE and DI rats. CRF given to LE and DI rats at 0.4 micrograms/100 g BW elicited a larger increase in ACTH plasma values than a similar dose of AII, 5 or 15 min after the injection. Moreover, ACTH levels after CRF in DI rats were significantly greater than those obtained in LE controls. In vitro studies using dispersed anterior pituitary cells indicate that the response of cells from either LE or DI rats to AII or AVP (both at 10(-9) and 10(-8)M) was similar. Cells from DI donors were hyperresponsive to CRF (2 X 10(-11) and 10(-10)M) in terms of ACTH release when compared with the response of cells from LE rats. The present results suggest that the presence of AVP is not essential to mediate the central response to AII and that AII may act centrally to stimulate CRF release from the hypothalamus in vivo, which would then enhance ACTH output. The results in the DI rat indicate that the increased response to CRF may be an important compensatory mechanism involved in the regulation of adrenocortical function in the DI rat.     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

Effects of water deprivation on corticosterone production in the male Brattleboro rat genetically deprived of vasopressin

Effects of 72 h water-deprivation on plasma corticosterone concentration have been investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI) and in male Long-Evans rats (LE), as controls. To determine the global effect of water deprivation, drinking water deprived rats were compared with hydrated animals. Because water deprived rats showed a depressed food intake, to elucidate the specific effect of dehydration alone, drinking water deprived rats were compared with similar food-restricted but water supplied animals. Increases in adrenal weights and in plasma corticosterone content, following 72 h water-deprivation, were greater in DI than in LE rats. In LE rats, they seemed to be the result of both dehydration and denutrition. Conversely in DI rats lacking vasopressin, dehydration alone increased neither adrenal weights nor plasma concentration of corticosterone; the whole plasma corticosterone content was reduced. So, in DI rats, the global response to drinking water deprivation was essentially due to food restriction, whose effect was partly suppressed by dehydration. Whatever the circumstances, plasma concentrations of corticosterone were higher in DI than in LE rats. Interrelationships between water deprivation, stress, vasopressin and glucocorticoids are discussed.     

Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

Natriuretic effect of atrial natriuretic peptide in diabetes insipidus rats

Washout of the solute concentration gradient in the renal medullary interstitium has been suggested to play a role in mediating the natriuretic response to atrial natriuretic peptide (ANP). The purpose of this study was to determine the effects of ANP 8-33 on sodium excretion in Brattleboro diabetes insipidus (DI) rats, in which medullary tonicity is known to be decreased as compared to Long-Evans (LE) control rats. Basal urine osmolality (Uosm) was significantly lower in DI rats as compared to LE rats (123 +/- 6 vs 673 +/- 38 mOsm/kg). Infusion of ANP 8-33 at a rate of 4 micrograms/kg/hr for 60 min resulted in a significantly greater increase in UnaV (delta 6.1 +/- 1.2 vs delta 2.9 +/- 0.7 microEq/min) and urine flow (delta 40 +/- 12 vs delta 8 +/- 7 microliter/min) in the LE rats than in the DI rats. The greater natriuresis occurred in the LE rats despite no significant change in Uosm. Fractional lithium reabsorption (an indicator of proximal sodium reabsorption) decreased similarly in both groups. Infusion of ANP had no effect on mean arterial pressure in LE and DI groups. In summary, infusion of ANP in the DI rat resulted in a significant natriuresis, albeit less than in LE rats. The natriuresis in the LE rats occurred despite no significant change in Uosm. These data suggest that mechanisms other than medullary washout are responsible for the natriuretic effects of ANP.     

Effects of vasopressin replacement during food-restriction stress

The effects of subcutaneous injections of vasopressin in vasopressin-deficient (Brattleboro or DI) rats were observed during nonstress (habituation) and stress (food-restriction) conditions as compared to other rats. Four groups of animals were employed: 1) Long-Evans (LE) rats that were food restricted with no injections (normal control animals), 2) DI rats that were food restricted with no injections, 3) DI rats injected with vasopressin, and 4) DI rats injected with peanut oil (vehicle). The parameters studied were: body weight, food intake, water intake, and gastric ulcer formation. With respect to body weight, water intake, and ulcer formation, two sets of animals emerged. The vasopressin-injected DI rats resembled the LE control rats, whereas the peanut oil-injected DI rats were similar to the DI rats with no injections. The former set of animals showed a higher body weight, reduced water intake, and fewer gastric ulcers than the latter set of animals. Thus the vasopressin-injected DI rats and the LE control rats could cope with the stress of food restriction, but the peanut oil-injected DI rats and the DI rats with no injections could not.     

Effects of naloxone and cholecystokinin on food and water intake in vasopressin-deficient rats (Brattleboro strain)

Opioid peptides and cholecystokinin (CCK) have been shown to play a role in regulation of feeding behavior. Another neuropeptide that has recently been suggested to be involved in feeding is vasopressin. We explored possible interactions between opiates, CCK and vasopressin in feeding regulation by studying feeding suppression produced by naloxone and CCK in Brattleboro (DI) rats, which are homozygous for diabetes insipidus and lack the ability to synthesize vasopressin. Ten DI and 15 age-matched Long Evans (LE) rats were food deprived for 14 hours on two different days and then injected with naloxone (2.5 mg/kg) on one day or saline on the other. Thirty minutes later the food was returned and food and water consumption were measured after 1, 3 and 4 hr. Naloxone suppressed the food consumption of both DI and LE rats but the suppression was greater for the DI rats. This result was specific to feeding as water consumption was suppressed in LE more than in DI rats. Two weeks later, the same rats were food deprived for 6 hours on two different days and then injected with CCK-8 (2.5 micrograms/kg) on one day and with saline on the other. Food was returned one minute after the injection and food and water consumption were measured 30 and 60 minutes later. Food intake was reduced equally for both DI and LE rats. Water intake was not reduced. The results suggest that the suppression of feeding by CCK does not require an intact vasopressinergic system. The greater feeding suppression by naloxone in DI rats may suggest that opiates are interacting with vasopressin in producing their effects on food intake.     

FMRF-NH2-like peptide is deficient in the pituitary gland of the Brattleboro rat

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2), isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. In the rat, F-8-F-NH2 immunoreactivity (IR) is highly localized in the neurohypophysis. In this study, F-8-F-NH2-IR was studied in the hypothalamo-neurohypophyseal system of an Arg8-vasopressin (AVP)-deficient animal, the Brattleboro (DI) rat, and the normal control Long-Evans (LE) strain. F-8-F-NH2-IR in the DI pituitary is below the level of detection in contrast to that in the LE (0.50 +/- 0.04 pmol/gland). Neuropeptide Y (NPY) levels are increased two-fold in the DI pituitary while AVP levels are below detection. The content of F-8-F-NH2-IR in the hypothalami and spinal cords of DI and LE rats is not statistically different, suggesting that the absence of F-8-F-NH2-IR in the Brattleboro pituitary is not due to a genetic defect in F-8-F-NH2 biosynthesis. The results of this study raise the question whether AVP could be involved in the regulation of F-8-F-NH2 immunoreactivity in the neurohypophysis.     

Atrial natriuretic factor (ANF) gene expression in the Brattleboro rat

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone of cardiac origin. It has natriuretic, diuretic and vasorelaxant properties and inhibits several cardiovascular modulators. Because of the possible effects of arginine vasopressin (AVP) on ANF secretion, we have investigated ANF gene expression in Brattleboro rats which are genetically deficient in AVP. Our results indicate that cardiac ANF mRNA and ANF content are higher in Brattleboro rats compared to Long-Evans controls, whereas the plasma levels are similar in both groups. Typical secretory granules containing immunoreactive ANF are present in ventricular cardiocytes of Brattleboro but not of Long-Evans rats. These data suggest that ANF release may be uncoupled from its synthesis in the absence of AVP.     

Inhibition by antiserum to rat growth hormone-releasing factor of growth hormone secretion induced by a met5-enkephalin analog, FK33-824, in rats

A Met5-enkephalin analog, FK33-824 (5, 10 and 20 micrograms/100 g body wt, iv) caused a dose-related increase in plasma growth hormone (GH) in urethane-anesthetized male rats. Pretreatment with cysteamine (30 mg/100 g body wt, sc), a depletor of hypothalamic somatostatin, increased the plasma GH response to FK33-824 (10 micrograms/100 g body wt, iv). Antiserum specific for rat GH-releasing factor (GRF) (0.5 ml/rat, iv) blunted GH release induced by FK33-824 (10 micrograms/100 g body wt, iv) in rats with or without cysteamine pretreatment. These results suggest that GH secretion induced by the opioid peptide is mediated, at least in part, by hypothalamic GRF in the rat.     

Effects of atrial natriuretic factor on blood pressure and the renin-angiotensin-aldosterone system

Atrial natriuretic factor (ANF) antagonizes vasoconstriction induced by numerous smooth muscle agonists and also lowers blood pressure in intact animals. ANF has particularly marked relaxant effects on angiotensin II-contracted vessels in vitro. Sensitivity to the blood pressure-lowering effect of ANF in vivo appears to be enhanced in renin-dependent models of renovascular hypertension compared with other experimental hypertensive models. The depressor action of low, possibly physiological doses of ANF in two-kidney, one-clip Goldblatt rats is due to a decrease in total peripheral resistance. On the other hand, high doses of ANF can lower cardiac output, particularly in volume-expanded models such as deoxycorticosterone-salt hypertension. ANF markedly inhibits renin secretion in intact animals, probably via increased glomerular filtration rate and load of sodium chloride to the macula densa. This effect is masked when renal perfusion is impaired (e.g., via unilateral renal artery constriction), in which case ANF may stimulate renin secretion slightly. ANF also reduces plasma aldosterone in vivo and inhibits basal and agonist-induced aldosterone release from isolated adrenal cortical cells. This effect appears to be especially marked for angiotensin-induced aldosterone production in vivo and in vitro. These findings indicate that ANF has potentially important interactions with the renin-angiotensin-aldosterone system and suggest a role for ANF in the homeostatic control of blood pressure as well as of extracellular fluid volume.     

Effects of adrenalectomy and hypercorticism on the ACTH content of the anterior and posterior pituitary in rats with inherited diabetes insipidus (Brattleboro strain).

The effects of adrenalectomy (Adx) and hypercorticism on the ACTH content in the anterior (AH) and the neurointermediate lobe (NIL) of the pituitary in Long Evans (+/+), heterozygous (+/DI) and homozygous (DI/DI) Brattleboro rats were determined using dispersed adrenal cells bioassay. Adx decreased the NIL-ACTH content in +/DI and DI/DI rats and left it unchanged in the +/+ rats. Adx increased the AH-ACTH content in the three groups. Hypercorticism had a delayed decreasing effect both in the AH and in the NIL in all rats, with one exception for the NIL in DI/DI rats. Conversely to what appeared in Wistar rats, in Long Evans and Brattleboro rats the corticosterone administered in drinking water was unable to reduce the increase in AH-ACTH activity. These data suggest that Brattleboro, and, to a lesser extent, Long Evans rats from which the former are derived present some particularities in the regulation of their corticotropic function at the AH and the NIL level. We also observed that NaCl (0.9%) added to drinking water and hypercorticism are two factors able to increase diabetes insipidus in homozygous rats without modifying the water intake in Long Evans and heterozygous rats.     

Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography

A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-stimulated aldosterone secretion. IC50 values for ANF were approximately 320 pM. The protocol that was established consisted of extraction of rat atria in 5 N acetic acid containing protease inhibitors. The extract was lyophilized, resolubilized in 0.1% trifluoroacetic acid containing 1% (w/v) sodium chloride, and subjected to RP-HPLC. Extraction of small batches of atria (i.e., from 10 or 20 rats) resulted generally in a yield of 2 nmol per rat (i.e., approximately 30 micrograms). The identity and purity of the pro-ANF were confirmed by the determination of both the amino acid composition and the amino-terminal sequence. Purified pro-ANF was radioiodinated and the efficiency of the extraction and purification procedure was assessed by adding labeled peptide to the initial tissue extract. The structural integrity and overall recovery of radioactivity were determined by RP-HPLC. The purification scheme provides undamaged pro-ANF of high purity. Purified pro-ANF was compared with synthetic rat ANF in the rat adrenal glomerulosa cell and isolated rat aortic strip bioassays. The peptides were apparently equally active in the adrenal cell system and approximately threefold less potent in relaxing aortic strips. The apparent equipotency in the adrenal cell bioassay may be due to the conversion of pro-ANF to ANF-like peptides during the bioassay incubation.     

Identification of aldosterone secretion inhibitory factor in bovine adrenal medulla

We report the first demonstration of an Aldosterone Secretion Inhibitory Factor (ASIF) in acid extracts of bovine adrenal medulla. Following separation from catecholamines and enkephalins, this factor leads to an 80% inhibition of PGE1-stimulated secretion of aldosterone from bovine adrenal zona glomerulosa. ASIF is retained on cation exchange gels and behaves as a small 5K-dalton peptide on Sephadex G-50. This factor cross-reacts in a radio-receptor assay for [125I] atrial natriuretic factor (ANF). ASIF is distinct from all neuropeptides formerly detected in the adrenal medulla, e.g. somatostatin, enkephalin, neuropeptide Y, dynorphin, neurotensin. In the adrenal gland, this ANF-like factor is predominantly found in the medulla (4 pmol/mg protein), with only trace amounts in the cortex (0.1 pmol/mg protein). ASIF might perhaps correspond to the endogenous ligand for the receptor sites that we have previously identified with [125I]ANF in bovine adrenal cortex and could contribute to the formerly reported attenuating influence of the adrenal medulla on mineralocorticoid production.     

Vasopressin deficiency and circadian rhythms during food-restriction stress

Vasopressin-containing, Long-Evans (LE) rats and vasopressin-deficient, Brattleboro (DI) rats were monitored for activity and core body temperature via telemetry. Rats were exposed to a 12-12 light-dark cycle and allowed to habituate with ad lib access to food and water. The habituation period was followed by an experimental period of 23 h of food-restriction stress in which a 1-h feeding period was provided during the light cycle. Although both strains of animals showed nocturnal activity and temperature rhythms during the habituation period, DI rats were more active than LE rats. The DI rats also had a lower body temperature in the dark. During the experimental period, both strains exhibited a phase shift of activity and body temperature correlating with the presentation of food. The DI rats developed a diurnal shift more rapidly than LE rats. The DI animals showed a dramatic increase in activity during the light phase and a marked decrease in body temperature during the dark phase. The LE animals showed a significant attenuation of activity, but maintained both nocturnal and diurnal temperature peaks throughout the food-restricted condition.