Homozygous Brattleboro Research Articles

Presence and synthesis of vasotocin in the pineal gland of Brattleboro rats.

Institute of Endocrinology and Faculty of Medicine, Bucharest, Romania (Received 20 September 1977) The pineal gland of all mammals so far investigated contains arginine-vasotocin (AVT; Pavel & Petrescu, 1966) or lysine-vasotocin (Pavel, 1965), except that of the homozygous Brattleboro rat with hereditary hypothalamic diabetes insipidus, in which apparently no radioimmunoassayable vasotocin could be detected (Rosenbloom & Fisher, 1975). Since it has been demonstrated recently that the pineal glands of normal rat foetuses contain (Pavel, Goldstein & Calb, 1975) and synthesize (Pavel, Goldstein, Ghinea & Calb, 1977) relatively large amounts of AVT, we investigated the possible occurrence and synthesis of AVT in the pineal glands of foetal homozygous Brattleboro rats. We also studied the pineal glands of adult homozygous Brattleboro rats to test for the presence of AVT. The methods of assay used have been described previously (Pavel, 1965) and included rat antidiuretic, rat uterine and frog (Rana temporaria) bladder assays.

The Effects of Adrenalectomy and Glucocorticoid Replacement on Vasopressin and Vasopressin-Neurophysin in the Zona Externa of the Median Eminence of the Rat

Adrenal regulation of the vasopressincontaining fibers to the portal capillary system in the zona externa of the median eminence was studied in the rat. Tissue sections of the hypothalamus were examined by immunoperoxidase technique and light microscopy using antisera to oxytocin and arginine vasopressin, and an antiserum which reacts with all rat neurophysins. These peptides were localized in the intact normal rat and after adrenalectomy, glucocorticoid replacement with dexamethasone, and after dehydration. The effect of adrenalectomy was also examined in the homozygous Brattleboro rat with diabetes insipidus (DI rat) which lacks arginine vasopressin and, arginine vasopressinneurophysin. In the zona externa of the normal rat a small number of arginine vasopressin and neurophysin- containing fibers, and an occasional one containing oxytocin were traced to the portal capillary bed. The zona externa of the unoperated DI rat contained only an occasional neurophysin- and oxytocin-reactive fiber. In the normal...

The fetal hypothalamus and pituitary during growth and differentiation

In our project on the involvement of the fetal brain and pituitary in intrauterine growth and parturition, data on human anencephalics are compared with those obtained in experiments with rat fetuses. An intrauterine growth spurt was observed in intact rat fetuses (starting at day 19 of pregnancy) and normal human fetuses (around 20 weeks of pregnancy), which appeared to be dependent on the integrity of the fetal brain and pituitary. Rat experiments showed that the acceleration of intrauterine growth was caused by endogenous fetal α-melanocyte-stimulating hormone (α-MSH). This hormone is also shown to be present in normal human fetal pituitaries during the intrauterine growth spurt. This, together with the absence of α-MSH fluorescence in anencephalic pituitaries suggests the involvement of α-MSH also in human fetal growth. The normal course of labour appeared also to be dependent on the integrity of the fetal brain and pituitary in man and rat. Since a protracted course of labour was found in Brattleboro rats being homozygous for diabetes insipidus, the fetal hypothalamo-neurohypophyseal system was thought to stimulate the course of labour. However, using immunofluorescence techniques, that enabled the specific localization of oxytocin and vasopressin in the adult rat, these hormones were not observed in the pituitary of fetal Wistar rats near term. Instead, a cross reacting compound which was probably arginine-vasotocin (AVT) was found throughout the hypothalamo-neurohypophyseal system which, by immuno-electronmicroscopy, appeared to be localized within the neurosecretory vesicles of the fetal neurohypophysis. The presence of AVT was confirmed by radioimmunoassay. AVT was absent or only present in very small quantities in the pituitary of homozygous Brattleboro fetuses. AVT from the fetus may play a role in the course of normal delivery. The presence of AVT in the fetal rat neurohypophysis and of α-MSH in the intermediate lobe of the fetal human pituitary shows, that fetal endocrine systems might be different from those, operating in adult organisms.

Hypertension in rats with hereditary diabetes insipidus. The role of age.

The effect of age, sex and salt intake on the hypertension produced in homozygous (DI) and heterozygous (non-DI) Brattleboro rats and Long Evans rats was studied. The left kidney was removed at the age of 25, 35, 50 and 80 days (UNX 25, UNX 35, UNX 50, UNX 80), and 0.6% NaCl solution or water was offered as drinking fluid. In UNX DI rats drinking saline the mean value of blood pressure (BP) exceeded 150 torr. The highest values of BP were observed in DI UNX 25 females while no hypertension occurred in rats UNX 80. There was no correlation between individual values of BP and saline consumption in DI females with hypertension. However, individual BP values correlated with the urinary Na+/K+ ratio measured in the course of 24 h of water deprivation, due to age-dependent sodium excretion the values of which were highest in the UNX 25 group. Hydronephrosis was present in all DI rats with manifest hypertension. In hypertensive animals, BP values, Na+/K+ ratio and the frequency of hydronephrosis exhibited the same age dependence. The role of age and adaptability to the increased saline intake in the susceptibility of DI rats to experimental hypertension is discussed.

Vasopressin contamination as a cause of some apparent renal actions of prolactin

The injection or infusion of NIAMDD prolactin (NIH P-S-10) into unanesthetized rats resulted in water and electrolyte retention with a large increase in urine osmolality but no effect on glomerular filtration rate. Since these effects on urine output were also observed in homozygous Brattleboro rats, the antidiuretic activity could not have caused by the release of endogenous antidiuretic hormone. Radioimmunoassay of NIH prolactin showed that it was contaminated with vasopressin (20 ng/mg of prolactin). By comparison, Sigma prolactin had no observed effect on urine excretion and contained very little vasopressin (2.5 ng/mg). It is concluded that some of the renal effects of prolactin have been reported in the literature may have been caused by the contaminating vasopressin.

Identification, in the external region of the rat median eminence, of separate neurophysin-vasopressin and neurophysin-oxytocin containing nerve fibres.

Immuno-enzyme cytochemical investigations, using single and double staining techniques, showed that the external region of the rat median eminence contains separate neurophysin-vasopressin fibres and neurophysin-oxytocin fibres. These neurophysin-hormone containing nerve fibres are influenced by bilateral adrenalectomy and by colchicine treatment. The external region of the median eminence of the homozygous Brattleboro rat contains neurophysin-oxytocin fibres. It does not contain immuno-reactive neurophysin-vasopressin fibres. Bilateral adrenalectomy also influences the neurophysin-vasopressin containing neurons of the suprachiasmatic nuclei. In the neurons of the parvicellular part of the rat hypothalamic paraventricular nuclei, staining for vasopressin and for oxytocin is completely absent.

Immuno-cytochemical demonstration of the inability of the homozygous Brattleboro rat to synthesize vasopressin and vasopressin-associated neurophysin.

Immuno-enzyme cytochemical investigations have shown that, (1) the hypothalamic supraoptic and paraventricular nuclei of the Brattleboro rat, as in the normal rat, contain separate neurons which produce oxytocin + neurophysin; (2) the hereditary inability of the Brattleboro rat to synthesize vasopressin and its associated neurophysin is due to a biochemical defect of separate "neurophysin-vasopressin" neurons in the supraoptic and the paraventricular nuclei. These observations strongly support the hypotheses that (1) vasopressin and its associated neurophysin are formed via a common precursor, and (2) the initial point of intracellular appearance of the hereditary defect in the Brattleboro rat lies in the synthesis of this precursor, which occurs on ribosomes. Moreover, observations have demonstrated that, in the Brattleboro rat, in addition to the hereditary inability of the hypothalamic magnocellular neurosecretory system to synthesize vasopressin, there also exists a similar hereditary defect in the hypothetical parvicellular suprachiasmatic-median eminence neurosecretory system.

Vasopressin and oxytocin content of microdissected hypothalamic areas in rats with hereditary diabetes insipidus.

Vasopressin was virtually absent from 5 microdissected hypothalamic areas and from the posterior pituitary glands of homozygous Brattleboro rats. The oxytocin concentration was normal in all these areas except for the arcuate nucleus, where it was absent. These results support the concept that Brattleboro rats have a specific defect in biosynthesis of vasopressin. The significance of the absence of oxytocin from the arcuate nucleus in these rats remains to be determined.

Specificity of oxytocin and vasopressin immunofluorescence.

A total inhibition of immunofluorescence could not be obtained using the technique of preincubating either anti-vasopressin or anti-oxytocin plasmas with their homologous antigens. An alternative test of specificity was therefore developed. Lysine-vasopressin (LVP), arginine-vasopressin (AVP) or oxytocin were covalently bound to CNB-activated agarose beads. These hormone-coupled beads were then either placed immediately on glass slides and treated in the same way as tissue sections for immunofluorescence, or pre-incubated in test-tubes with the antibodies and then prepared for immunofluorescence. Fluorescence was measured quantitatively using a Leitz orthoplan microscope with epi-illumination and a photometer attachment. Without pre-incubation the anti-oxytocin plasmas produced intensive fluorescence on those beads containing their homologous antigen (oxytocin) but only slight fluorescence with the heterologous antigens (AVP or LVP). Anti-vasopressin plasmas produced intensive fluorescence of AVP-, LVP- and oxytocin-containing beads. Because the neurohypophysial hormones were bound to agarose beads, antibodies binding to these hormones could be removed by simple centrifugation. Anti-oxytocin and anti-vasopressin lost their fluorescence capacity after pre-incubation with oxytocin- or vasopressin-containing beads respectively, showing that all the antibodies important for fluorescence were bound to homologous antigens. Pre-incubation of anti-oxytocin or anti-vasopressin with beads coupled to their heterologous hormones completely removed the components binding to the heterologous hormone, leaving antibodies that showed fluorescence only with oxytocin or vasopressin respectively. This purification showed that in contrast to the findings with homologous antigens, not all of the antibody population bound to its heterologous antigen. Using non-purified anti-vasopressin, immunofluorescence was observed in neurohypophyses of homozygous Brattleboro rats. This was evidently due to those antibodies which bind to oxytocin, since the fluorescence was abolished after pre-incubating the antibody-containing plasmas with oxytocin-coupled beads. Immunofluorescence was still observed, however, in the suprachiasmatic nucleus of both Wistar and heterozygous Brattleboro rats, if purified anti-vasopressin was used. This was probably due to either vasopressin storage or production in these hypothalamic cells.

Changes in electrolyte excretion following intraperitoneal injection of dibutyryl cyclic AMP in the brattleboro rat

Intraperitoneal injection of 40 μg of dibutyryl cyclic AMP to homozygous Brattleboro rats fasted for 12 hours but having free access to water resulted in an increase in urine flow and in the rates of excretion of Na, K, PO 4 and urea. Similar results were obtained in Brattleboro rats having free access to food and water which received 200 μg of nucleotide. The injection of 20 μg of dibutyryl cyclic AMP or 40 μg 5′-AMP to homozygous Brattleboro rats with free access to food and water or the administration of 40 μg of 5′-AMP to homozygous rats fasted for 12 hours but with free access to water did not alter renal excretion patterns. These results indicate that dibutyryl cyclic AMP may depress tubular reabsorption of water, Na and PO 4 as has been shown to be the case in the intact dog. In homozygous Brattleboro rats fasted for 24 hours but allowed free access to water, 40 μg of dibutyryl cyclic AMP resulted in a tendency to reduced urine flow and diminished excretion of Na, K and urea. The explanation for these results is not apparent from these data.

Changes in organ weights and blood parameters in ageing Brattleboro rats with hereditary diabetes insipidus.

Organ weights and blood parameters were measured in male and female Brattleboro rats with hereditary diabetes insipidus (DI) and in normal rats of the same strain, between 37 and 138 weeks old. Although differences in body weight between the homozygous DI and heterozygous normal rats accounted for differences in the weights of the heart and gonads, the weights of the spleen, liver and kidney were not solely body-weight dependent; there were significant sex and genotype differences in the mean weights of these organs. The liver, heart and gonads were the only organs showing age-dependent changes. No genotype-dependent differences were observed in the haematocrit and haemoglobin concentration, although there was a sex difference; the mean values of these parameters were higher in males. Plasma glucose concentrations were significantly lower in homozygous than in heterozygous rats; in the latter, plasma glucose concentration decreased with age. No significant changes occurred in the mean plasma concentrations of sodium or potassium with age or genotype. Total plasma lipids of heterozygous female rats showed both age- and genotype-dependent changes; this parameter reached a plateau at 90-100 weeks old, and was significantly higher in heterozygous females than in all other groups. Plasma proteins showed no age-dependent changes, but were of higher mean value in heterozygous than in homozygous rats and were greater in females than in males. Differences in blood parameters of homozygous and heterozygous Brattleboro rats are discussed in terms of differences in hormone concentrations.

Stimulation of ADH release by the renin-angiotensin system.

Stimulation of ADH release by the renin-angiotensin system.