Hilda Weyl Sokol

THE HORMONAL STATUS OF THE BRATTLEBORO RAT

Although the primary defect in the homozygous (DI) Brattleboro rat is the inability to synthesize vasopressin, other endocrine abnormalities are manifested in this animal as well. It is believed that such differences from the normal condition are probably secondary effects attributable to the lack of vasopressin and/or the state of diabetes insipidus itself. Replacement therapy with exogenous vasopressin corrects the high fluid turnover in these rats as well as some, but apparently not all. of the accompanying abnormalities. The purpose of this paper is to review what is currently known or surmised ?.bout the hormonal status of this valuable experimental model. Unless its endocrine and other physiological deviations are known and taken into account, the experimental results obtained with this animal could be misleading.

Genetic approaches to the study of the regulation and actions of vasopressin.

Publisher Summary This chapter describes two animal models with hereditary diabetes insipidus (D.I.): (1) rats with hereditary hypothalamic D.I. and (2) mice with hereditary nephrogenic D.I. These two models have been useful in investigating major problems in renal and endocrine function and disease. The chapter discusses how these two models helped to elucidate questions in three major areas: (1) the production of neurohypophysial hormones and their carrier proteins, the neurophysins, (2) salt and water balance in some disease states, and (3) the cellular mode of action of vasopressin. Considerable evidence favoring the one neuron-one hormone hypothesis has been obtained in D.I. rats that cannot produce vasopressin but can produce oxytocin. At both light- and electron-microscopic levels, the supraoptic and paraventricular nuclei of these rats reveal two populations of neurons: one containing neurosecretory material and the other not. These findings have been supported by immunohistological studies in D.I. rats that show one group of perikarya reacting with antibody to neurophysin and another group of perikarya that does not. Furthermore, the presence of many reactive and nonreactive cells in both the supraoptic and paraventricular nuclei strongly suggests that oxytocin and vasopressin are produced in both magnocellular nuclei. The growth of D.I. rats is retarded when compared to normal rats or to heterozygotes of the Brattleboro strain that produce an intermediate amount of vasopressin. The deficient growth can be largely corrected by exogenous growth hormone that does not influence the high rate of fluid turnover.

Evidence for Oxytocin Synthesis After Electrolytic Destruction of the Paraventricular Nucleus in Rats with Hereditary Hypothalamic Diabetes Insipidus

The reduced amount of neurosecretory material (NSM) that is stored in pituitary glands of hereditary hypothalamic diabetes insipidus (DI) rats compared with glands of normal rats reflects their oxytoc

Studies of anterior pituitary-grafted rats: I. Abnormal prolactin response to thyrotropin releasing hormone, clonidine, insulin, and fasting

Although the rat implanted with extra anterior pituitary glands (AP) under the kidney capsule has been widely used as a model of chronic hyperprolactinemia, its hormonal status has not been fully characterized. Using conscious, unrestrained female pituitary-grafted rats and sham-operated littermates, we investigated prolactin (PRL) secretion in response to the following stimuli: thyrotropin releasing hormone (TRH), clonidine, insulin, and fasting. The AP-implanted rats had a greater and more sustained rise in serum PRL after TRH than control rats, reflecting a direct effect of TRH on the ectopic lactotropes. In contrast after clonidine, which acts via the hypothalamus, the serum PRL rose to much higher levels in sham-operated rats than in rats bearing ectopic pituitary tissue. Both insulin-induced hypoglycemia and fasting decreased serum PRL in control rats, but the AP-implanted animals manifested a rise in serum PRL in response to these stimuli. Thus, the AP-implanted rat is not only a valid model of excess and abnormal PRL secretion, but it may also be useful for distinguishing between stimuli requiring an intact hypothalamic-pituitary unit and agents which act directly on the pituitary gland.

Studies of anterior pituitary-grafted rats: II. Normal growth hormone secretion

Of the various animal models used to study chronic hyperprolactinemia, the otherwise intact rat implanted with extra anterior pituitary glands (AP) under the kidney capsule is assumed to be normal except for excess circulating prolactin (PRL). Since the ectopic glands contain numerous somatotropes in addition to abundant and active lactotropes, it was important to assess growth hormone (GH) secretion as well in this model of hyperprolactinemia. The structural and functional similarities of PRL and GH are such that it is necessary to demonstrate that metabolic abnormalities noted in AP-implanted rats are due to hyperprolactinemia and not to altered GH secretion. AP-implanted female rats have significantly higher resting serum PRL concentrations when compared to sham-operated control rats, but baseline serum GH levels are similar in normal and pituitary-grafted rats. Suppression of GH by insulin and clonidine is comparable in AP-implanted and control rats. The intrasellar pituitary GH concentration is also similar (ca. 20 micrograms/mg wet weight) in hyperprolactinemic and normal rats. We conclude that GH secretion is normal in the non-hypophysectomized AP-implanted rat, in contrast to the hypophysectomized AP-implanted rat model which has been reported to have diminished GH secretion. Despite the presence of recognizable somatotropes, the ectopic anterior pituitary does not appear to secrete significant amounts of GH, making the intact rat bearing multiple pituitary grafts an excellent model of chronic hyperprolactinemia.

The hypothalamohypophyseal neurosecretory system in mice with vasopressin-resistant urinary concENTRATING DEFECTS.

Abstract Three distinct genotypes of mice with vasopressin-resistant urinary concentrating defects are compared to a normal strain with respect to urine volume and osmolality, serum osmolality, and sizes of supraoptic (SON) and paraventricular (PVN) neurons and the various parts of the pituitary gland. Mice with severe diabetes insipidus (DI) produce large volumes of dilute urine; their hypertrophied SON and PVN cells have markedly enlarged nucleoli; the pars nervosa (which stores ample amounts of neurosecretory material), the pars distalis and the pars intermedia are all larger than normal, the latter exhibiting the greatest relative increase in size. The two mouse strains with “mild DI” occupy intermediate positions in most of the above parameters suggesting an interrelationship between the amount of fluid turnover and the degree of hypertrophy. Additionally, the results suggest that there may be a relationship between increased neurohypophyseal activity and the hypertrophy of the pars intermedia.

Demonstration that Rat Prolactin Has No Intrinsic Antidiuretic Activity in the Rat

The proposition that rat prolactin has an intrinsic antidiuretic activity was examined using a conscious rat model (Brattleboro homozygotes, DI rats) which does not appear to produce arginine vasopressin (AVP). A preparation of rat prolactin obtained from NIAMDD, rat prolactin B1, produced a prompt antidiuresis when administered intravenously to these animals. Prolactin B1 was found to contain 150 ng of AVP/mg protein by RIA. All of the measurable AVP was removed from prolactin B1 by polyacrylamide gel electrophoresis and the electrophoresed prolactin was completely devoid of any antidiuretic activity. Addition of synthetic AVP to electrophoresed prolactin in an amount equivalent to the AVP contamination of prolactin B1 completely restored the originally observed antidiuretic activity. Synthetic AVP given alone produced a similar antidiuresis, but in the presence of electrophoresed prolactin these effects were produced by much smaller amounts of AVP. We believe that our data demonstrate that rat prolactin has no intrinsic antidiuretic activity, and that the antidiuretic activity associated with different prolactin preparations could be entirely due to their contamination with AVP. The data also show that the presence of prolactin enhanced by about five times the antidiuretic activity of AVP.

PROLACTIN AND GROWTH HORMONE IN THE UNSTRESSED BRATTLEBORO RAT

Because Brattleboro (DI) rats are smaller than aged-matched Long-Evans (LE) rats, we investigated anterior pituitary function in unstressed, unanesthetized rats fitted with indwelling intra-aortic catheters. Male DI and weightmatched LE rats received intra-arterial injections of thyrotropin-releasing hormone (TRH) (400 ng/100 g BW), insulin (1 unit/100 g BW), or the alpha-adrenergic agent, clonidine (0.1 mgl100 g BW), at approximately weekly intervals. Blood samples for prolactin (PRL), growth hormone (GH) , and glucose (in the insulin test) were obtained through the catheter just before and at specific intervals after the injection. Serum separated from whole blood was frozen for later radioimmunoassay using reagents kindly provided by the National Institute of Allergy, Metabolism, and Digestive Diseases (National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases) Hormone Distribution Program. At sacrifice, anterior pituitaries were removed, and halves were frozen for later radioimmunoassay of GH and PRL concentration or preserved in Bouin’s solution for immunocytochemical analysis. The frozen halves were thawed, weighed, and homogenized in phosphosaline buffer just before assay. Pituitary glands from female homozygous (DI) , heterozygous (HZ), and Long-Evans (LE) rats as well as male HZ rats were prepared at the same time as the preserved hemi-pituitaries from this study. Transverse 6 pm sections of Bouin’sfixed, paraffin-imbedded glands were incubated with the same antisera to rPRL and rGH as used in the radioimmunoassay of these hormones. Subsequent application of peroxidase-antiperoxidase and enzyme-substrate 1-3 resulted in the deposition of brown reaction products at specific antigenic sites.

Altered Catecholamine Innervation of the Supraoptic Nucleus in the Nephrogenic Diabetes insipidus Mouse

Fluorescence histochemical and immunocytochemical techniques were used to investigate morphologic correlates of the relationship between catecholamine varicosities and vasopressin-containing perikarya in an animal model of vasopressin excess, the nephrogenic diabetes insipidus mouse. Our results show hypertrophy and increased immunoreactivity in vasopressin neurons in these mice were accompanied by a marked increase in the density and to some extent the fluorescence intensity of catecholamine varicosities within the supraoptic nucleus. These results further support the concept of functional interactions between catecholamine and vasopressin neurons and raise the possibility that the target neuron, or one of its products, perhaps vasopressin, either exerts a trophic influence on the catecholamine innervation pattern of the supraoptic nucleus or enhances catecholamine content in existing fibers and terminals.

DECREASED SENSITIVITY TO OXYTOCIN OF UTERI FROM HOMOZYGOUS BRATTLEBORO RATS

Although most female homozygous diabetes insipidus (DI) rats of our Brattleboro rat colony can become pregnant, litters are smaller and somewhat less viable. This may be due to three factors: (a ) DI rats experience difficulties during delivery, (b) stillborns occur more frequently, and (c) mothers often destroy their pups. Difficult parturition could result from inadequate myometrial stimulation by oxytocin (OT) . We have tested the possibility that uteri from DI rats are less sensitive to OT and that lack of vasopressin can influence uterine responses. The results of these experiments are reported here.