Immunocytochemical staining of supraoptic neurons from homozygous Brattleboro rats by use of antibodies against two domains of the mutated vasopressin precursor

Summary By use of an antibody against the 14 amino acids in the mutated vasopressin precursor (CP-14) characteristic of the homozygous Brattleboro rat, an immunohisto- and-cytochemical study was performed on the supraoptic nuclei of homozygous Brattleboro rats. At the light-microscopic level, varying numbers of perikarya per section exhibited a positive reaction. The most intense staining was observed in a patchy manner on the peripheral portions of the cytoplasm, its central portion being stained less intensely. The antiserum did not react with the supraoptic perikarya of the Wistar rat. In the homozygous Brattleboro rat, antibodies against normal vasopressin only rarely resulted in a positive immunoreaction. However, when it was observed, incubation of the subsequent section with CP-14-antiserum suggested a co-localization of both peptides in the same perikaryon. At the ultrastructural level, CP-14 immunoreactivity was demonstrated on the secretory cisternae of the Golgi apparatus, on lysosome-like bodies and on parts of the rough endoplasmic reticulum. With the use of an antibody against normal vasopressin, immunoreactivity was confined to very limited areas of the rough endoplasmic reticulum. The oxytocin immunoreactivity in supraoptic perikarya of Brattleboro rats did not differ from that in the Wistar rat, either at the light- or at the electron-microscopic levels.     

Oxytocin as well as vasopressin potentiate ovine CRF in vitro

We have investigated the effects of synthetic oxytocin and vasopressin on corticotropin release induced by the 41-residue ovine corticotropin-releasing factor (oCRF) in vitro. Segments of the anterior pituitary glands obtained from male and female Wistar or from female hetero- and homozygous Brattleboro rats were used. Ovine CRF (0.1-2.5 nmol/l) stimulated corticotropin release by pituitaries of Wistar rats and this effect was augmented two- to threefold in the presence of arginine vasopressin (0.09-0.9 mIU/ml) or oxytocin (0.9-90 mIU/ml). A similar phenomenon was demonstrated in Brattleboro rats. These data favor the hypothesis that oxytocin might have a physiological role in the regulation of pituitary-adrenocortical function in homozygous Brattleboro rats which lack vasopressin.     

A comparison of the febrile responses of the Brattleboro and Sprague-Dawley strains of rats to endotoxin and endogenous pyrogens

The febrile responses of homozygous (di/di) Brattleboro rats, to both intravenous endogenous pyrogen and to a lipopolysaccharide endotoxin, were compared with those of normal Sprague-Dawley rats. There were no detectable differences between the fever curves of the two strains in response to endogenous pyrogen. Brattleboro rats, which are deficient in the neuropeptide arginine vasopressin (AVP), displayed fevers that were both qualitatively and quantitatively indistinguishable from those of normal Sprague-Dawley rats that do not suffer from congenital diabetes insipidus. It is concluded that the absence of AVP-containing cells in Brattleboro rats is not an important factor in determining the nature of their febrile responses to endogenous pyrogen. More remarkable, however, were the divergent febrile responses of the two strains to intravenously injected endotoxin. Normal rats displayed hypothermic responses, whereas the Brattleboro rats became febrile. By 2 h after the injection of endotoxin, body temperatures in both strains had returned to normal. Three hours after the rats had been exposed to endotoxin, both strains were found to be totally refractory to endogenous pyrogen. However, when both strains of rats were tested to endogenous pyrogen 3 days later, their febrile responses were more than double the magnitude of their initial control responses. These alterations in the febrile responsiveness of rats occurring at different times after the injection of endotoxin appear to be related to the effects that endotoxin has on the cells of the reticuloendothelial system, over the same time course.     

High-Protein Carboxymethylase Activity and Low Endogenous Methyl Acceptor Proteins in Posterior Pituitary Lobe of Rats Lacking Neurophysin-Vasopressin (Brattleboro Rats)

Abstract: 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.     

Possible influence of tachykinins on body fluid homeostasis in the rat

The effect on water intake, urine flow and vasopressin release of intracranial injections of substance P, physalaemin and eledoisin was studied in Wistar and Brattleboro, homozygous and heterozygous, rats. The tachykinins strongly inhibited water intake both in Wistar and in Brattleboro, homozygous and heterozygous, rats. Physalaemin and eledoisin reduced urine flow in Wistar and heterozygous, but not in homozygous, Brattleboro rats. Substance P never affected urine elimination. Physalaemin and eledoisin produced a dose-dependent, long lasting release of vasopressin in Wistar rats. Substance P did not affect the release of vasopressin. The results suggest that both substance P and physalaemin could influence brain mechanisms which control water intake, acting as thirst inhibitors, and that physalaemin could also participate in body fluid control by conserving water through vasopressin release.     

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.     

Effect of vasopressin gene expression on the growth of Walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WA rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG x Brattleboro) F1 hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG x Brattleboro) F1 rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.     

Age-dependent DOCA-salt hypertension in Brattleboro rats: the role of vasopressin

The age-dependent participation of endogenous vasopressin (VP) during the development of DOCA-salt hypertension was studied in young (28-day-old) and adult (75-day-old) Brattleboro rats. VP-deficient homozygous (DI) rats were compared to heterozygous (non-DI) littermates which do synthetize VP. Six weeks of DOCA-salt treatment did not increase blood pressure (BP) in adult DI rats. On the other hand, in young DI animals there was a significant rise of systolic and mean arterial pressure accompanied by the hypertrophy of the left ventricle. This moderate DOCA-salt hypertension of young DI rats contrasted with severe hypertension of young non-DI rats. Increased BP response of young VP-deficient DOCA-salt treated rats was independent of the saline intake or blood volume expansion which were similar in young hypertensive and adult normotensive DI animals. It could be concluded that vasopressin is not essential for the induction of DOCA-salt hypertension in young rats even if VP is responsible for the magnitude of BP elevation. In contrast to young animals vasopressin is very important for the development of DOCA-salt hypertension in adult rats.     

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat

Summary CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats.CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.     

Effect of vasopressin gene expression on the growth of walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WAG rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG × Brattleboro) F₁ hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG × Brattleboro) F₁ rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.     

Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol

We have tested the hypothesis that animals with reduced levels of arginine vasopressin (AVP) would show reduced tolerance to ethanol. Brattleboro rats either heterozygous or homozygous for the diabetes insipidus (DI) trait and normal Sprague-Dawley rats were exposed to ethanol vapor for 21 days. Two days later, tolerance was evaluated by monitoring body temperature reductions after intraperitoneal injection of 2 g/kg (20% w/v) ethanol. Under the same conditions of chronic ethanol exposure, Sprague-Dawley rats, but not Brattleboro rats, displayed tolerance to the hypothermic effects of intraperitoneal ethanol. This phenomenon did not appear to be related to differences in ethanol metabolism or blood alcohol levels in Brattleboro rats. These data support a possible role for AVP in the development or maintenance of tolerance.     

Regional analysis of brain opiate receptors in rats with hereditary hypothalamic diabetes insipidus.

The regional distribution of stereospecific ³H-dihydromorphine binding sites in Brattleboro rats with hereditary hypothalamic diabetes insipidus was studied. Control animals were homozygous normal Brattleboro rats and rats heterozygous for the vasopressin deficiency. Scatchard plots of ³H-dihydromorphine binding in a washed membrane preparation showed that rats with diabetes insipidus exhibited higher receptor concentrations in all assayed areas of the cerebral hemispheres. In the diencephalon, receptor concentrations were lower in diabetes insipidus rats. The results point to the existence of interactions between brain opioid systems and neurohypophyseal peptides.     

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.     

Vasopressin gene expression is attenuated in the fetal Brattleboro rat

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.     

Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.     

Disorders of cell acquisition in the brain of rats deficient in vasopressin (Brattleboro mutant)

Neural cell acquisition and certain features of its underlying mechanisms were determined in the forebrain, cerebellum and olfactory bulbs of Brattleboro rats which were either heterozygous (control) or homozygous for a hereditary deficiency in vasopressin synthesis. In comparison with heterozygotes, the amount of DNA, which provides a measure of total cell number, was decreased in homozygous animals. The magnitude of the effect varied markedly from one brain part to another. Although more [³H]thymidine was retained in homozygous animals, the corrected values for DNA labeling showed a reduction in comparison with heterozygous rats. This decrease in mitotic activity preceded the reduction in brain cell number. The observed disturbances in brain cell acquisition of the Brattleboro mutant indicate a possible role for vasopressin in early brain development.     

Vasopressin stimulates endocytosis in kidney collecting duct principal cells

In target epithelia, a vasopressin-induced water permeability increase is accompanied by the appearance of intramembranous particle (IMP) clusters, probably representing water-permeable patches, in the apical plasma membrane of responding cells. In the collecting duct principal cell, we have previously shown that these clusters are located in clathrin-coated pits. To determine whether vasopressin induces the endocytic uptake of these membrane domains in principal cells, we have examined the uptake of horseradish peroxidase (HRP) by principal cells of normal rats, vasopressin-deficient Brattleboro rats, and vasopressin-treated Brattleboro rats, following intravenous injection of HRP. By quantitative electron microscopy, principal cells of Brattleboro homozygous rats were found to take up much less HRP into cytoplasmic vesicles than normal rats, and HRP uptake was increased to normal levels in vasopressin-treated Brattleboro rats. Many invaginating coated pits at the cell surface were loaded with HRP reaction product, indicating their participation in the observed endocytosis of HRP. We conclude that vasopressin stimulates endocytosis in collecting duct principal cells. Since we have already shown that IMP clusters are found in coated pits at the cell surface, the endocytic removal of these putative water-permeable patches from the apical membrane seems to occur via a clathrin-mediated mechanism in this tissue.     

Enhanced release of a ‘prostacyclin-like’ substance from aortic strips of spontaneously hypertensive rats

The release of an endogenous ‘prostacyclin-like’ substance from aortic strips of 8 male Wistar rats of the New Zealand genetically hypertensive strain (GH) was compared with that of 8 weight, age and sex matched normotensive Wistar control rats. The amount of ‘prostacyclin-like’ substance released by the aortic strips into tris buffer, under the influence of mechanical stimulation, was measured by its ability to inhibit human platelet aggregation as compared to the inhibitory effect of standard prostacyclin sodium salt. It was shown that generation of this substance increased with incubation time and that a significantly greater amount was produced by GH rats.     

Hypothalamo-posterior pituitary system in Brattleboro rats: immunoreactive levels of leucine-enkephalin, dynorphin (1-17), dynorphin (1-8) and alpha-neo-endorphin

The levels of immunoreactive leucine-enkephalin, alpha-neo-endorphin, dynorphin (1-17) and dynorphin (1-8) have been determined in the hypothalamus and posterior pituitary from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous (producing vasopressin) for diabetes insipidus, and from male and female Long Evans rats. In the hypothalamus we found no significant differences in the levels of these peptides while there were great differences in extracts from the posterior pituitary: female homozygous animals have greatly reduced levels in all four peptides compared to the heterozygous controls. In male homozygous animals the differences in the dynorphin (1-17) and leucine-enkephalin levels were small whereas the concentrations of alpha-neo-endorphin and dynorphin (1-8) showed a significant decrease compared to the male heterozygous controls. The results indicate a reduction in opioid peptides linked to the vasopressin deficiency in a partially sex dependent manner.     

Effect of the di mutation (non-sugar diabetes) on properties of kidney proteins in rats

Renal proteins were studied in Brattleboro rats of the didi genotype and in Wistar and Sprague-Dawley rats with normal alleles +2 of this loci. In animals of the +2 genotype maintained under conditions of water deprivation, the content of 120 kDa protein increased significantly in inner medulla of the kidney over 3 days. No changes in the amount of this protein were observed in rats of the didi genotype under the same conditions. We suggest that the congenital inability of didi mutants to synthesize vasopressin accounts for the distinctions observed in the reactions of rats with different genotypes.