Homozygous Brattleboro Research Articles

Co-existence of unrelated peptides in oxytocin and vasopressin terminals of rat neurohypophyses: Immunoreactive methionine 5-enkephalin-, leucine 5- enkephalin- and cholecystokinin-like substances

In neurohypophyses of normal rats and of vasopressin-deficient Brattleboro rats, applying immunocytochemical methods, we have found (1) in oxytocin terminals co-existing methionine-enkephalin-, cholecystokinin- and possibly leucine-enkephalin-like substances and (2) in vasopressin terminals, co-existing leucine-enkephalin-like material. The correspondence between the enkephalin immunoreactivity patterns and the oxytocin or vasopressin immunoreactivity pattern in serial 0.5 μ thick sections was so close that occurrence of enkephalin in separate nerves appears unlikely. At a subcellular level the enkephalin- and the cholecystokinin-like immunoreactivities were localized mainly in the neurosecretory granules that also contain oxytocin or vasopressin; there was no evidence for separate enkephalingranule populations. The intensity and frequency of leucine-enkephalin immunostaining in vasopressin terminals was much enhanced by treatment of the deplasticized sections with trypsin prior to incubation with antibodies. This suggests incorporation of the leucine-enkephalin sequence into longer peptide chains, presumably dynorphin and/or α-neo-endorphin. In oxytocin endings tryptic cleavage enhanced cholecystokinin-like immunoreactivity, while methionine-enkephalin immunostaining was as intense without enzyme treatment. The opioid peptide forms of vasopressin neurons appear to be elongated chains containing exclusively the leucine-enkephalin sequence, whereas in oxytocin neurons methionine-enkephalin as free pentapeptide seems to prevail. Association of enkephalins with vasopressin and oxytocin was also indicated when methionine- and leucine-enkephalin contents of neurointermediate lobes of homozygous Brattleboro rats were compared with peptide stores in heterozygous controls in a high pressure liquid chromatography system and highly specific leucine- and methionine-enkephalin radioimmunoassays. We assume that the opioid peptides in the oxytocin system and in the vasopressin system are derived from different opioid precursor molecules.

High-protein carboxymethylase activity and low endogenous methyl acceptor proteins in posterior pituitary lobe of rats lacking neurophysin-vasopressin (Brattleboro rats).

The activity of protein carboxymethylase and the endogenous protein methyl acceptor capacity were examined in the posterior, intermediate, and anterior lobes of the pituitaries of homozygous Brattleboro rats with diabetes insipidus and in heterozygous Brattleboro and Long-Evans control rats. Protein carboxyl methylation is selectively altered in the posterior pituitary lobes of homozygous Brattleboro rats. Protein carboxymethylase activity is higher (+40%) and endogenous methyl acceptor protein capacity is lower (-80%) with respect to heterozygous Brattleboro and Long-Evans control rats. This latter change is correlated with decreased methylation of proteins of a molecular weight of approximately 11K daltons, is selective for the posterior pituitary lobe, since it does not occur in the intermediate and anterior lobes, and probably reflects the absence of vasopressin-associated neurophysin in homozygous Brattleboro rats. Our results support a physiological role of protein carboxyl methylation in the neurosecretory process in the posterior pituitary gland.

Vasopressin-immunoreactive cell bodies in the bed nucleus of the stria terminalis of the rat.

In the dorsal and ventral portions of the bed nucleus of the stria terminalis of the rat numerous cell bodies immunoreactive for vasopressin and neurophysin II were found after colchicin pretreatment. These cells are predominantly multipolar but sometimes also bipolar, and have a width and length of approximately 9 and 16 microns, respectively. In the homozygous Brattleboro rat, which is deficient in vasopressin, no immunoreactive vasopressin was found in these cells. Following incubation with anti-oxytocin and anti-bovine neurophysin I, only magnocellular immunoreactive cell bodies were found in the septal region. The consequences of these results concerning the vasopressin fiber pathways in the brain are discussed.

Excretion of 6-keto-PGF 1α and PGE 2 in rats with hereditary hypothalamic diabetes insipidus

We measured the urinary excretion of a stable metabolite of prostacyclin, 6-keto-PGF 1α, and of PGE 2 in homozygous Brattleboro rats, a strain with hereditary hypothalamic diabetes insipidus. Excretion of both prostaglandins was largely increased compared to both heterozygous Brattleboro rats and Long-Evans rats. These results are in contrast to previously published observations demonstrating a subnormal excretion of PGE 2 in Brattleboro rats. It is suggested that prostaglandin synthesis may be under tonic inhibitory control by vasopressin both in the kidney and in the endothelial cells of blood vessels. The findings further support the view that prostaglandins play an important role in the regulation of water excretion and in the pathogenesis of polyuric conditions.

Levels of pro-neo-endorphin/dynorphin-derived peptides in the hypothalamo-posterior pituitary system of male and female Brattleboro rats

Immunoreactivities for leucine-enkephalin and the related opioid peptides α-Neo-endorphin, dynorphin(1–17) and dynorphin(1–) were measured in hypothalamus and poterior-intermediate pituitary extracts from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous(able to produce vasopressin) for daibetes insipidus. In hypothalamus no differences were found in peptide levels among the 4 groups of animals. In contrast, striking, but variable differences were found in posterior intermediate pituitary. In homozygous male animals dynorphin(1–17) immunoreactivity was not significantly different from levels measured in heterozygous male animals. Immunoreactivities for the 3 other peptides, however, showed greatly reduced levels in homozygous male animals compared to heterozygous male animals. Female homozygous animals had greatly reduced levels of all 4 peptides (including dynorphin(1–17) compared to female heterozygous controls. In addition, female heterozygous animals had considerably higher levels of all peptides than male heterozygous animals. In contrast, no sex differences were found in normal Long-Evans rats of the strain from which Brattleboro rats were derived. The following conclusions could be drawn from these findings. (1) Homozygous Brattleboro rats have reduced levels of Leuenkephalin-related opioid peptides in posterior pituitary due to defect or alteration of secretion or turnover but dot due to a defect in biosynthesis. (2) This defect is partially sex dependent and is directly or indirectly linked to the vasopressin deficiency. (3) Since normal rats do not show sex differences in peptides levels, the partial sex dependence of the opioid peptide defect in posterior pituitary seems to be X-chromosomally linked to the vasopressin deficiency.

Estrogen-concentrating neurophysin-containing hypothalamic magnocellular neurons in the vasopressin-deficient (Brattleboro) rat: a study combining steroid autoradiography and immunocytochemistry

This report describes the distribution of neurophysin-containing, estradiol-concentrating neurons in a strain of rat which is congenitally unable to produce vasopressin and its associated neurophysin (the Brattleboro rat). In this strain of rat, all of the neurophysin-containing cells are oxytocin producing. The magnocellular neurons which produce vasopressin in the normal rat are present in their normal numbers and normal locations (Rhodes, C. H., J. I. Morrell, and D. W. Pfaff (1981) J. Comp. Neurol. 198: 45-64) and can be identified as the neurophysin-negative magnocellular neurons. Estradiol-concentrating cell nuclei were observed in magnocellular neurons with neurophysincontaining cytoplasm as well as in magnocellular neurons lacking immunocytochemically detectable neurophysin. The majority of these neurons were found in the paraventricular nucleus (PVN), ventral and medial to its lateral subnucleus, and in the posterior subnucleus of the PVN. There were, in addition, many neurophysin-containing and neurophysin-lacking magnocellular neurons with nuclei which did not concentrate estradiol. Within the PVN, the majority of the neurophysinnegative, non-estradiol-concentrating neurons were in the lateral subnucleus, while the majority of the neurophysin-positive, non-estradiol-concentrating neurons were in the medial subnucleus. Comparison of the results of experiments using homozygous Brattleboro rats with the results of similar experiments using the (normal) parent strain Long-Evans rat suggests that, in the normal animals, there are both oxytocinand vasopressin-producing neurons which concentrate estradiol. Comparison of these observations with published descriptions of the anatomical distribution of neurons which project to the medulla or spinal cord suggests that many of the oxytocin.or vasopressin-containing, estrogen-concentrating neurons in the PVN send axons to regions regulating autonomic functions. Estrogen treatment is a stimulus for the release of oxytocin (Yamaguchi et al., 1979) and vasopressin (Skowsky et al., 1979). A decrease in immunocytochem’ This paper is dedicated to the Rev. Dr. Winthrop Brainerd, Rector of Christ’s Church, Baltimore, formerly Keeper of Books and Manuscripts of Her Majesty’s College of Heralds, and author of Codici Monastici Ethiopiani. We wish to thank IL A. Zimmerman for the anti-neurophysin antiserum used in this work. This work was supported in part by National Institutes of Health Grants HD 05751 and HD 16327. C. H. R. was the recipient of National Research Service Award 5T32GM07739. ’ Present address: Department of Pathology, Hospital of the University of Pennsylvania, Philadelphia, PA 19104. ” To whom correspondence should be addressed at Rockefeller University, 1230 York Avenue, New York, NY 10021. ically identified oxytocin has been observed in the anterior commissural nucleus (ACN) in response to estrogen treatment (Rhodes et al., 1981c). It is not known whether other magnocellular cell groups also participate in the estrogen-stimulated increase in plasma oxytocin levels or where estrogen acts to produce this effect. The fall in pituitary oxytocin content during proestrus and estrus in normal cycling female rats observed by Crowley et al. (1978) suggests that physiological changes in ovarian steroid levels affect the release of posterior pituitary peptides. The combination of steroid autoradiography and immunocytochemistry has been used to identify neurophysin-containing, estradiol-concentrating neurons in the (normal) parent strain Long-Evans rat (Rhodes et al., 1981a). That work was done with a primary antiserum

Brain Receptors for Blood-Borne Calcitonin in Rats: Circumventricular Localization and Vasopressin-Resistant Deficiency in Hereditary Diabetes Insipidus*

Specific binding sites for blood-borne calcitonin were localized by means of quantitative radioautography to the circumventricular organs of the rat brain. By this method, using normal Long-Evans rats as controls, specific binding of blood-borne calcitonin in the median eminence region of the hypothalamus was reduced by one-third in homozygous Brattleboro rats, which are genetically deficient in vasopressin. Competitive binding analysis in vitro of the hypothalami from these animals confirmed the binding deficit in homozygous rats, and Scatchard analysis suggested a reduction in the number of binding sites. In homozygous rats daily vasopressin replacement therapy restored normal water balance but did not normalize the hypothalamic calcitonin binding deficit. These studies delineate for the first time specific sites within the central nervous system which could serve to mediate direct actions of blood-borne calcitonin on brain function. The deficit in the Brattleboro rat may provide a model for further investigation of the role of calcitonin within selective regions of the central nervous system.

Oral antidiuretic therapy: studies in the diabetes insipidus rat.

In hypothalamic diabetes insipidus, water balance is achieved primarily through the thirst mechanism. The administration of an antidiuretic agent in the drinking water should restore the antidiuretic response to volume and osmoregulatory drive. To test this hypothesis, homozygous Brattleboro strain rats were given arginine vasopressin (AVP) or 1-desamino-8-D-arginine vasopressin (dDAVP) in the drinking water in concentrations of 10-10,000 micrograms/l (AVP) and 5-10,000 micrograms/l (dDAVP). Oral AVP was found to be ineffective. Oral dDAVP resulted in 1) a progressive increase in dDAVP dose, from 2.3 to 2,559 micrograms.day-1.kg-1; 2) a dose-dependent increase in urine osmolality from 306 to 1,796 mosmol/kg; and 3) a dose-dependent decrease in urinary solute excretion. At each dDAVP dose level, stable physiological states were achieved within 24 h. Similar antidiuretic states were achieved when dDAVP was administered in increasing doses or when therapy was initiated at a high dose. These findings demonstrate that inclusion of an appropriate antidiuretic agent in the drinking water can restore the renal response to volume-osmoregulatory drive.

Correlation between water intake and dendritic branching in homozygous Brattleboro rats: A Golgi study

This report is the fourth in a series on the brains of three groups of male Brattleboro rats. The present study concerns only homozygous Brattleboro rats. One group raised in standard environmental conditions was killed at 60 days of age, another raised in standard conditions was killed at 90 days of age, and a third group raised in standard conditions for 60 days was killed at 90 days of age after 30 days of environmental enrichment. Water intake (measured between 33 and 40 days of age) was compared with the number of branching segments on basal dendrites of pyramidal neurons in the upper layers of the parietal and occipital cortices. A significant negative correlation between water intake and the number of dendritic branching segments per primary branch was found in both parietal and occipital cortices in the 90-day-old standard condition group. In contrast, the 90-day-old enriched group showed a significant positive correlation between water intake and segments per primary branch in the parietal cortex, and a similar but nonsignificant response to enrichment in the occipital cortex. These results may reflect a cumulative effect of the congenital absence of vasopressin which is apparent by 90 days of age and which is modified by environmental enrichment. In the light of our previous data linking vasopressin and dendritic branching, we speculate that the neuropeptide, oxytocin, may be a physiologic correlate linking the behavioral parameter (water intake) and the anatomic parameter (dendritic branching) in homozygous Brattleboro rats.

The Carboxy Terminus of the Precursor to Vasopressin and Neurophysin: Immunocytochemistry in Rat Brain

A pituitary glycopeptide whose amino acid sequence was previously identified has now been recognized as the final portion of the precursor to arginine vasopressin and its associated neurophysin. Immunocytochemical techniques with antiserums against this 39 amino acid peptide and vasopressin were used to study their distribution in the rat central nervous system. The peptide is located in vasopressin-synthesizing cells in the neurosecretory magnocellular nuclei. Positively stained fibers project from the magnocellular nuclei through the median eminence to the posterior pituitary. Studies of the homozygous Brattleboro rat, which is known to be deficient in the production of vasopressin and its related neurophysin, also show the absence of immunoreactivity to this peptide. These immunocytochemical data strongly indicate that the peptide is synthesized with vasopressin.

Vasopressin-reversible increase in angiotensin-converting enzyme in specific hypothalamic nuclei of Brattleboro rats

The activity of the angiotensin-converting enzyme (ACE) (kininase II, EC 3.4.15.1) was examined in 5 discrete hypothalamic nuclei of rats lacking vasopressin (homozygous Brattleboro rats, DI, di/di)and their corresponding controls (heterozygous Brattleboro rats, HZ, di/+, and Long Evans, LE, +/+ rats), with and without hormonal replacement with arginine-vasopressin (AVP). DI rats showed a vasopressin-reversible increased ACE activity when compared with LE controls, HZ rats showing intermediate activity. These changes occurred only in the supraoptic and periventricular hypothalamic nuclei, and were absent in other hypothalamic areas studied, including the paraventricular nucleus. These results provide biochemical evidence in support of previous anatomical and physiological data, for an interaction between the brain vasopressin and angiotensin systems in discrete hypothalamic nuclei, and suggest that vasopressin could regulate the formation of brain angiotensin II by modulating the activity of the converting enzyme.

Membrane retrieval by vacuoles after exocytosis in the neural lobe of Brattleboro rats

The possible role of microvesicles and vacuoles in the recapture of membrane after pituitary hormone release by exocytosis has been studied in homozygous Brattleboro rats. These mutant animals are unable to synthesize vasopressin and exhibit a steady state hypersecretion of oxytocin from the neural lobe as a result of the osmotic imbalance caused by their diabetes insipidus. This can be converted to a second steady state which approximates to the rate of secretion found in normal Long Evans rats by the administration of exogenous vasopressin daily for 30 days. In the Brattleboro rat, presumptive oxytocinergic nerve endings contain typical 160–170 nm diameter neurosecretory granules; other magnocellular nerve endings contain a population of smaller ( ~ 100 nm diameter) dense-cored granules. The number of dense-cored granules was reduced in both types of nerve ending in the hypersecreting Brattleboro rats, but increased as a result of vasopressin treatment to levels which, for the classical neurosecretory granules, approximated that found in Long Evans rats. The microvesicle population of the nerve endings was essentially similar in quantitative terms in all the three groups (i.e. hypersecreting Brattleboro rats; vasopressin-treated Brattleboro rats and Long Evans controls). The number of vacuoles, on the other hand, was increased in nerve endings in the hypersecreting animals but reduced to levels found in Long Evans rats in the Brattleboro animals treated with vasopressin. Furthermore, the size of the vacuoles was comparable to the size of the dense-cored granules contained in the nerve endings. These changes in the vacuole population are exactly those that would be predicted for an organelle responsible for recapture of the granule membrane. We threfore conclude that membrane retrieval after exocytosis of neurosecretory granules in the neural lobe is achieved by vacuoles and that these organelles probably retrieve the membrane of the granule intact.

Oxytocin action: lipid metabolism in adipocytes from homozygous diabetes insipidus rats (Brattleboro strain).

Oxytocin, like insulin, stimulates glucose oxidation in normal rat adipocytes. Fat cells from homozygous Brattleboro rats that exhibit diabetes insipidus (HoDI animals) and that have a normal number of oxytocin receptors, however, are unable to respond to oxytocin in terms of glucose oxidation. We now report that in adipocytes from HoDI animals that are responsive to insulin, oxytocin was also unable to stimulate lipogenesis. In contrast, oxytocin like insulin was able to inhibit epinephrine-stimulated lipolysis in adipocytes from HoDI animals. Thus, in HoDI adipocytes, the results indicate that the receptor-effector system is only partially defective.

Role of vasopressin in hypertension: studies using the Brattleboro rat

Plasma arginine vasopressin (AVP) levels were elevated in both one-kidney, one-clip [1K-1C) and two-kidney, one-clip (2K-1C) hypertensive Long-Evans rats. Homozygous Brattleboro rats with hereditary hypothalamic diabetes insipidus (DI), which are completely devoid of vasopressin, were made hypertensive using the 1K-1C Goldblatt models for renal hypertension. The 2K-1C DI rats developed hypertension at the same rate and to the same degree as normal 2K-1C hypertensive Long-Evans rats. The development of hypertension in 1K-1C DI rats was similar to the normal 1K-1C hypertensive Long-Evans rats. However, the absolute levels of systolic blood pressure reached were significantly less in the vasopressin-deficient rats. Treatment with 1-desamino-8-D-arginine vasopressin (DDAVP), the synthetic analogue of arginine vasopressin that has high antidiuretic but low pressor potencies, was associated with restoration of water balance in the volume-depleted DI rats and also restored blood pressure to hypertensive levels equivalent to the 1K-1C Long-Evans rats. These studies suggest that vasopressin is not essential for the development of experimental renal hypertension in rats but may contribute to the absolute levels of systolic blood pressure reached through its properties as an antidiuretic hormone.

Vascular lesions and angiotensin in malignant hypertension in the absence of vasopressin.

1. Vasopressin deficient homozygous Brattleboro rats develop malignant renal hypertension following complete aortic-ligature between the renal arteries. 2. This form of hypertension is associated with high plasma renin activity (PRA) and plasma angiotensin II (AII) levels and a high incidence of the specific vascular lesions of fibrinoid necrosis in both Brattleboro and Long-Evans rats. 3. The levels of PRA and AII in the malignant hypertensive Brattleboro rats were not different from those in Long-Evans rats with malignant hypertension. 4. No compensation by the renin-angiotensin system therefore could be demonstrated for the lack of vasopressin in malignant hypertensive Brattleboro rats. 5. Vasopressin does not appear to be essential as a pressor hormone in the development of malignant renal hypertension and fibrinoid can occur in the absence of vasopressin.

Inability of oxytocin to activate pyruvate dehydrogenase in the Brattleboro rat.

Oxytocin has insulin-like activity in that it stimulates lipogenesis and increases pyruvate dehydrogenase activity. However, in adipocytes from homozygous Brattleboro rats oxytocin is incapable of stimulating lipogenesis or pyruvate dehydrogenase activity, although insulin stimulation of both processes is normal and the antilipolytic activity of oxytocin is normal. Thus, the Brattleboro rat provides a new genetic model for the study of oxytocin action, wherein recognition of the chemical mediator is partially defective.

Altered pituitary-thyroid function in the Brattleboro rat with diabetes insipidus.

To determine the roles of vasopressin in the regulation of the pituitary-thyroid axis, we studied this axis in vasopressin-deficient Brattleboro rats with diabetes insipidus. When plasma TSH, T3, and T4 concentrations were determined by RIA, it was found that plasma TSH levels were increased by 100–170%, and T3 levels were lowered by about 20% in the homozygous (DI) and heterozygous (HZ) Brattleboro rats. The plasma T4 level decreased by about 20% in DI rats, whereas its level in HZ rats was similar to the control level. TRH (250 ng/ 100 g BW, iv) increased plasma TSH levels at 10 and 20 min in all three groups, but the TSH response to TRH in DI and HZ rats was significantly greater than that in the control rats. Continuous administration of desamino-D-arginine vasopressin (48 ng/day for 7 days, sc) normalized urine volume and osmolality in DI rats, but did not affect the abnormalities in the pituitary-thyroid axis. The same dose of DDAVP in HZ anials significantly decreased urine volume and increased osm...

Vasopressin: An essential pressor factor for blood pressure recovery following hemorrhage

Two experimental approaches were used to evaluate the importance of the pressor effects of vasopressin in blood pressure recovery following hypotensive hemorrhage. Experiments using homozygous Brattleboro rats demonstrated that the hemodynamic recovery of these animals was subnormal, even though the activation and efficacy of the sympathetic nervous and renin-angiotensin systems were intact. Experiments using an antipressor vasopressin analogue in normal rats during hypotensive hemorrhage demonstrated significantly blunted blood pressure recovery in the presence of the analogue. Thus, both experiments indicate that the pressor effects of circulating vasopressin play an essential role in blood pressure recovery following hypovolemic hypotension induced by hemorrhage.

Increased branching of basal dendrites on pyramidal neurons in the occipital cortex of homozygous Brattleboro rats in standard and enriched environmental conditions: A Golgi study

As a continuation of our previous reports in a series of studies on the brain of Brattleboro rats, the branching of basal dendrites of pyramidal neurons in the upper layers of the occipital cortex was quantified in three groups of male heterozygous and homozygous Brattleboro rats. One group raised in standard environmental conditions was killed at 60 days of age, and another from standard conditions was killed at 90 days of age. A third group from enriched environmental conditions was killed at 90 days of age after 30 days of enrichment. Comparing the two types of Brattleboro rats, the homozygous rats showed significantly more total dendritic branching segments per neuron in both the 60-day-old standard condition group and the 90-day-old enriched group. A similar measure (segments per primary branch) was also significantly greater in homozygous than in heterozygous rats at 60 days of age. In the 90-day-old enriched group, the homozygous rats showed a trend toward more segments per primary branch than the heterozygous rats. The results suggest that the complete absence of vasopressin produces metabolic effects which, at certain ages or in certain environmental conditions, increase the branching of basal dendrites of pyramidal neurons in the upper layers of the occipital cortex.

Dynorphin and vasopressin: common localization in magnocellular neurons.

The opioid peptide dynorphin is widely distributed in neuronal tissue of rats. By immunocytochemical methods, it was shown previously that dynorphin-like immunoreactivity is present in the posterior pituitary and the cells of the hypothalamic neurosecretory magnocellular nuclei which also are responsible for the synthesis of oxytocin, vasopressin, and their neurophysins. By using an affinity-purified antiserum to the non-enkephalin part of the dynorphin molecule it has now been demonstrated that dynorphin and vasopressin occur in the same hypothalamic cells of rats, whereas dynorphin and oxytocin occur in separate cells. Homozygous Brattleboro rats (deficient in vasopressin) have magnocellular neurons that contain dynorphin separate from oxytocin. Thus dynorphin and vasopressin, although they occur in the same cells, appear to be under separate genetic control and presumably arise from different precursors.