Leptin receptor expression and suppressor of cytokine signaling transcript levels in high-fat-fed rats.

Several lines of evidence suggest that obese individuals have a higher set point for body weight regulation relative to lean subjects. Since obese rodents and humans have high serum levels of leptin, it has been hypothesized that this may be the result of an insensitivity to this weight reducing hormone. In this experiment we assessed whether feeding of a high-fat diet to rats affects leptin receptor (OB-R) transcript levels or induces up-regulation of the suppressors of leptin/cytokine induced signaling, SOCS-3 and PIAS-3. We found that despite a significant weight gain associated with markedly increased circulating leptin levels neither OB-R gene expression nor SOCS-3 or PIAS-3 mRNA levels were significantly altered in the high-fat fed rats. This was in contrast to control experiments where administration of exogenous leptin induced a several-fold increase in SOCS-3. It is concluded that high-caloric food intake per se is not sufficient to provoke suppression of leptin signaling via these factors in animals without genetic predisposition to obesity.

Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities.

Besides an isomer of the cardenolide ouabain, a material with a similar HPLC retention time as ouabain but cross-reactivating with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump is known to circulate in human blood plasma (B. SICH et al., Hypertension 27, 1073-1078 (1996).). The concentrations of both substances are known to correlate with the blood pressure. It was the intention of this work to localize tissues that contain the highest concentrations of the proscillaridin A immunoreactive material, to correlate its concentration with that of ouabain and to get information whether the concentration of this material simply reflects the number of sodium pumps of the tissue extracted. Specific antibodies for each cardiotonic steroid were used to test the tissue concentration. This report shows that in bovine tissues the distribution pattern of proscillaridin A and ouabain immunoreactivities are similar and that hypothalamus and adrenals show the highest concentrations. The cross-reactive material did not reflect the number of sodium pumps per g of wet weight tissue as measured by [3H]ouabain binding. Therefore, it is unlikely that the tissue concentrations in both immunoreactivities reflects the tissue capacity of sodium pumps labeled with cardiotonic steroids via the blood plasma. The study rather favors the concept that two different types of inhibitors of the sodium pump exist within both tissues.

In situ hybridization demonstrates vasopressin gene transcription in hypothalamic neurons of crossbred hypertensive x diabetes insipidus rats.

In situ hybridization, Northern blotting, and solution hybridization assay were used to examine vasopressin-gene messenger ribonucleic acid (mRNA) transcripts in hypothalamic tissue from five strains of rats: Long-Evans, Wistar-Kyoto, and diabetes insipidus (Brattleboro) rats, spontaneously hypertensive-stroke-prone rats, and cross-bred diabetes insipidus x spontaneously hypertensive-stroke-prone rats. A 290 base-pair, single-stranded RNA probe, with 221 bases complementary to exon C of the vasopressin gene was synthesized by the SP6 transcription vector system. This probe labeled appropriate neurohypophysial hypothalamic neurons, as well as suprachiasmatic nucleus cells, in tissue samples from all five strains of rats. Confirming other recent hybridization results with diabetes insipidus rat brain tissue, diabetic animals were found to transcribe their mutated vasopressin gene. In addition, this investigation found that the cross-bred diabetic-hypertensive rat also synthesizes a vasopressin-gene messenger ribonucleic acid transcript. Quantitative analyses of solution and in situ hybridization results suggested the cross-bred diabetic-hypertensive rat exhibits a level of vasopressin-gene messenger ribonucleic acid similar to diabetes insipidus rats. This observation is consistent with previous data on the cross-bred diabetic-hypertensive rat which suggests they inherit the mutant vasopressin gene from Brattleboro rats.

Brain angiotensin II: localization and possible functions.

The results of biochemical, immunohistochemical and autoradiographic studies have led to the description of the endogenous brain angiotensin II system as a highly localized network of angiotensin II-containing cell bodies and fibers as well as characteristic sites with high angiotensin II receptor density. The specific localization of this system predominantly within limbic, hypothalamic and brain stem structures that are known to be involved in central cardiovascular regulation and the control of volume homeostasis suggests a possible functional role of brain angiotensin II in the control of these homeostatic functions. In particular, brain angiotensin II may function to help maintaining adequate tissue perfusion to vital parts of the body at times, when body fluid and cardiovascular homeostasis are jeopardized. In addition, there is evidence that brain angiotensin II may participate in the control of hormone release from the anterior and posterior pituitary gland, regulation of body temperature and the processing of learning contents. Future research may profitably be directed towards an exact functional characterization of central angiotensinergic pathways as well as an detailed analysis of the mechanisms that underly central angiotensin II-induced responses.

Distribution of neurophysin II immunoreactive nerve fibers within the subnuclei of the nucleus of the tractus solitarius of the rat.

The location of neurophysin II immunoreactive nerve fibers and preterminal processes has been examined in various functionally distinct subnuclei of the nucleus of the tractus solitarius (nTS) using the indirect immunofluorescence method for immunocytochemistry combined with cytoarchitectonic identification. The nTS is responsible for integrating respiratory and autonomic reflex activity: the vlnTS, vnTS, ni and nI are associated with respiratory activity; the dlnTS and dnTS are important sites for the integration of baroreceptor and chemoreceptor activity; the ncom, dnTS and dlnTS integrate cardiac afferent activity and the mnTS mediates both cardiovascular and gastrointestinal effects. At levels caudal to the obex, the ncom contained the largest number of neurophysin II immunoreactive nerve fibers and the mnTS and dmnX contained moderate neurophysin II immunoreactivity. At levels rostral to the obex the region of the dorsal medulla adjacent to the mnTS and dnTS (PVR and dPSR) showed the densest immunoreactivity and the mnTS, dmnX and vPSR showed moderate immunoreactivity. At the rostral pole of the nTS, neurophysin II immunoreactive nerve terminals were seen in the dendritic regions of cells in dmnX and mnTS. This selective distribution of neurophysin II immunoreactive nerve terminals in the cardiovascular and gastrointestinal subnuclei of the nTS implicates a direct, descending, hypothalamic, oxytocin-neurophysin II containing pathway interacting with these nTS functions. These results confirm the hypothesis (Sawchenko and Swanson) that descending neurophysin II immunoreactive pathways represent an important neuronal system for the hypothalamic regulation of cardiovascular (vasomotor) and gastrointestinal nuclei in the brainstem.

Development of a new strain of spontaneously hypertensive rats homozygous for hypothalamic diabetes insipidus.

The aim of the present study was to investigate whether the presence of arginine vasopressin (AVP) is necessary for the establishment of high blood pressure in spontaneously hypertensive rats (SHR). For this purpose we crossbred SHR of the stroke-prone substrain (SHRSP) with rats homozygous for hypothalamic diabetes insipidus of the Brattleboro strain (DI) which are unable to synthetize AVP. The successful introduction of the DI gene into the SHRSP strain (SHRDI) was demonstrated by the following observations: In 10-month-old rats, water intake was similarly elevated in SHRDI as in DI rats (137 +/- 6.5 vs 125 +/- 10.5 ml per 24 hours). AVP was undetectable in the plasma, in the hypothalamus, and in the pituitary of SHRDI and DI rats. Urine osmolality and urinary concentration of sodium and potassium were markedly reduced. SHRDI and DI did not adequately concentrate their urine during an 8-hour period of water deprivation, but both strains of rats responded well with a fall in urine output and a rise in urine osmolality to subcutaneous administration of the non-pressor analog of AVP, DDAVP. Mean arterial blood pressure was markedly increased in SHRDI as well as in SHRSP (184 +/- 9.7 vs 197 +/- 5.2 mm Hg). Thus, we have developed a new line of spontaneously hypertensive rats homozygous for hypothalamic diabetes insipidus. From this finding it is concluded that AVP is not essential for the development and maintenance of spontaneous hypertension of rats.

Differential regulation of beta-endorphin in the anterior pituitary, intermediate lobe, hypothalamus and brain stem.

The influence of adrenalectomy (adx), betamethasone (BET) and 11-deoxycorticosterone (DOC) on beta-endorphin-like-immunoactivity (beta-END-LI) in plasma, anterior (AP) and neuro-intermediate lobe (N-IL) of pituitary, hypothalamus (HT) and brain stem (BS) was studied in rats. One day or three weeks after adx animals were injected i.m. for five consecutive days with 0.4mg/kg BET or 3.4mg/kg DOC. Six days after adx beta-END-LI was increased in BS and plasma after four weeks there was an elevation in plasma and AP. BET suppressed beta-END-LI in plasma of both sham-adx and adx animals six days after operation and in the N-IL after six days of adx. DOC led to an increase of beta-END-LI in HT after sham-adx and to a reduction in the N-IL after four weeks of adx. These observations demonstrate that beta-END-LI is regulated differently at its sites of synthesis in the AP, N-IL and HT.

Effect of adrenalectomy and administration of hypertonic saline on the content of aldehyde fuchsin-positive neurosecretory material and posterior lobe hormones in the median eminence and the neural lobe of rats.

The influence of adrenalectomy and administration of hypertonic saline on the amount of vasopressin, oxytocin, and neurophysin contained in the median eminence and the neural lobe of rats was studied by means of the following methods: (i) morphometric and microphotometric analyses of aldehyde fuchsin-stained histological sections of the neurohypophysis; (ii) immunohistochemical demonstration of vasopressin, oxytocin, and neurophysin in the neurohypophysis, and (iii) radioimmunological measurement of vasopressin and oxytocin in extracts of the median eminence and the neural lobe. Adrenalectomy increases the amount of vasopressin and neurophysin in the external layer of the median eminence but does not change the content of oxytocin. It has no influence on the amount of vasopressin, oxytocin, and neurophysin demonstrable in the inner layer of the median eminence and in the neural lobe two weeks after the operation. Hypertonic saline markedly diminishes the vasopressin, oxytocin, and neurophysin content of the inner layer of the median eminence and the neural lobe but reduces only slightly, if at all, the amount of vasopressin and neurophysin in the outer layer of the median eminence. The findings support the concept that osmotic stress reduces only the vasopressin and oxytocin content of the hypothalamus-neural lobe system and has no or only little influence on the vasopressin content of the outer layer of the median eminence.

Distribution of neurophysins in rat brain: radioimmunological measurement and characterization.

Rat neurophysins were isolated from posterior pituitaries and antibodies raised in rabbits. Individual sensitive and specific radioimmunoassays for the neurophysins associated with either vasopressin or oxytocin were developed and employed for measuring the neurophysins in various regions of rat brain. Neurophysin-like immunoreactivity was detected in hypothalamus, septum, amygdala, brainstem and spinal cord. Characterization by high performance liquid chromatography suggested the identity with the neurophysins contained in the posterior pituitary. The distribution of these peptides in the brain points to a possible role in central nervous system processes.

Transmitter mediated arginine vasopressin release from superfused hypothalamus and pituitary gland.

The study was designed to investigate the effect of various neurotransmitters on the hypothalamus and pituitary gland to determine the sites of their action. Superfused isolated rat hypothalami and pituitary glands demonstrated basal secretion of arginine vasopressin (AVP) and repeated response to stimulation thus showing the viability of the preparation. Acetylcholine and histamine stimulated the release of AVP at the hypothalamic and pituitary levels; dopamine and norepinephrine released AVP in a dose related manner only from the hypothalamus; angiotensin II released AVP in the same fashion only from the pituitary gland. AVP secretion stimulated by dopamine and norepinephrine may represent synaptic inputs which are localized at the hypothalamus and must be distinguished from the site of action at the pituitary gland of angiotensin II.