Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30-40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

Impaired parathyroid hormone response to hypocalcemic stimuli in a patient with hypomagnesemic hypocalcemia.

Magnesium (Mg) deficiency sometimes causes hypocalcemia with impaired PTH secretion although the precise mechanism remains unclear. We examined the PTH secretion in response to physiological hypocalcemic stimuli in a patient with hypomagnesemic hypocalcemia. We adopted sodium bicarbonate infusion test, which we recently developed, to evaluate the PTH response to acute decrease in plasma ionized Ca. The results showed that, before Mg replacement and when the patient was mildly hypocalcemic, absolutely no PTH release to hypocalcemic stimuli was observed. In contrast, the plasma Ca was promptly normalized following the start of Mg replacement, and brisk PTH response to hypocalcemic stimuli was obtained during the same test carried out a week after the Mg replacement. The data in this case thus suggest that: a) the acute regulation of PTH release by plasma ionized Ca is lost in the patient with hypomagnesemic hypocalcemia, and b) Mg deficiency itself is likely to be a primary cause of this disorder because the hormone response was clearly restored after shortterm Mg replacement alone.

Overexpression of vasopressin in the rat transgenic for the metallothionein-vasopressin fusion gene.

Arginine vasopressin (AVP) is a major antidiuretic hormone, the overproduction of which causes diluting hyponatremia in humans and is called the syndrome of inappropriate antidiuresis (SIAD). To study physiological changes resulting from AVP overproduction and to develop an animal model of hyponatremia, the human AVP gene was expressed under the control of the metallothionein promoter in transgenic (Tg) rats. Analyses of AVP immunoreactivity (irAVP) in the tissues revealed that the transgene is expressed mainly in the central nervous system. Gel filtration showed that irAVP in the brain and plasma was properly processed AVP. AVP purified from the brains of both Tg and control rats also exerted equal bioactivity to generate cAMP in LLC-PK1 cells. The founder rats did not show any physical or anatomical abnormalities. Under basal conditions, Tg rats had high plasma AVP levels (Tg 13.8 +/- 2.5 pg/ml; control 2.7 +/- 1.2 pg/ml; n=6 in both groups; means +/- S.E.M.), decreased urine volume, and normal plasma [Na(+)]. Hypertonic saline injected i.p. did not affect AVP secretion in Tg rats. In response to a zinc-supplemented liquid diet, plasma AVP decreased in control rats, but increased in Tg rats (Tg 32.7 +/- 2.7 pg/ml; control 1.0+/-0.1 pg/ml; n=6), resulting in hyponatremia (Tg 135.2 +/- 2.5 mEq/l; control 140.8 +/- 0.4 mEq/l; n=6). To our knowledge, this is the first transgenic animal to show diluting hyponatremia. This transgenic rat may therefore provide a useful model in which to investigate various physiological alterations resulting from the oversecretion of AVP which involve SIAD, stress response, behavior, and blood pressure.

Effects of acute hypotensive stimuli on arginine vasopressin gene transcription in the rat hypothalamus.

We investigated the baroregulation of arginine vasopressin (AVP) gene transcription in the supraoptic (SON) and paraventricular nuclei (PVN) in conscious rats by use of intronic in situ hybridization. Hemorrhage of 16 ml/kg body wt decreased mean arterial pressure (MAP) by 57% and increased both plasma AVP (control, 1.2 +/- 0.3 pg/ml; 16 ml/kg body wt, 38.9 +/- 3.2 pg/ml) at 10 min and AVP heteronuclear (hn)RNA levels (SON, 150%; PVN, 140% of control values) at 20 min. On the other hand, hemorrhage of 7 ml/kg body wt had no significant effect on MAP, plasma AVP, or the AVP hnRNA levels. To better understand the baroregulation, we also examined the effects of sodium nitroprusside (SNP), which induces hypotension without a change in blood volume. The subcutaneous injection of 2 mg/kg body wt SNP, which decreased the MAP by 60%, increased both plasma AVP (control, 1.6 +/- 0.4 pg/ml; 2 mg/kg body wt, 8.1 +/- 0.4 pg/ml) at 10 min and AVP hnRNA levels (SON, 150%; PVN, 140% of control values) at 30 min. The injection of 0.1 mg/kg body wt SNP, which reduced the MAP by 10%, failed to increase either the plasma AVP or AVP hnRNA levels. These results indicate that AVP gene transcription increases rapidly after both hypotensive hemorrhage and normovolemic hypotension. In addition, it is suggested that the set point for AVP synthesis in the baroregulation is similar to that for AVP release.

Anorectic effect of pituitary adenylate cyclase activating polypeptide (PACAP) in rats: lack of evidence for involvement of hypothalamic neuropeptide gene expression.

We investigated the effect of centrally administered pituitary adenylate cyclase activating polypeptide (PACAP) on feeding in rats, and the involvement of hypothalamic neuropeptide gene expression using in situ hybridization. Intracerebroventricular injection of PACAP (1000 pmol/rat) significantly decreased food intake in a dose-dependent manner. In PACAP-treated rats, neuropeptide Y (NPY) mRNA levels in the arcuate nucleus and galanin mRNA levels in the paraventricular nucleus increased, and corticotropin-releasing hormone (CRH) mRNA levels in the paraventricular nucleus decreased. In rats fasted for 72 h, NPY mRNA levels increased, and CRH mRNA levels decreased, but galanin mRNA levels were unchanged. These results indicate that the anorectic function of PACAP is not mediated by NPY or CRH, and that PACAP increases galanin synthesis.

Centrally administered neuropeptide FF inhibits arginine vasopressin release in conscious rats.

There is evidence indicating that neuropeptide FF (NPFF) is an endogenous modulator of opioid systems. In the present study, we investigated the effect of centrally administered NPFF on arginine vasopressin (AVP) release in conscious rats. The plasma AVP increase in response to either hyperosmolality [i.p. injection of hypertonic saline (600 mosmol/kg)] or hypovolemia [i.p. injection of polyethylene glycol (PEG)] was significantly blunted when NPFF was injected into the lateral ventricle so that the given drug could act at the hypothalamus and also reach the brain stem (hypertonic saline with 10 micrograms/rat NPFF, 3.28 +/- 0.48 pg/ml; hypertonic saline alone, 7.85 +/- 1.78 pg/ml; PEG with 10 micrograms/rat NPFF, 4.07 +/- 1.40 pg/ml; PEG alone, 8.25 +/- 1.90 pg/ml). The plasma AVP increase in response to PEG-induced hypovolemia was also attenuated significantly and more potently when NPFF was injected into the cisterna magna so that the given drug could be readily accessible to the dorsal medulla where the nucleus of solitary tract is located (10 micrograms/rat; 2.71 +/- 0.14 pg/ml). In contrast, the NPFF injected into the cisterna magna had no significant effect on hyperosmolality-induced AVP release. Treatment with naloxone (10 mg/kg BW, sc) significantly reversed the inhibitory effects of NPFF on AVP release. These results suggest that central NPFF might play an inhibitory role via the hypothalamus in the osmoregulation of plasma AVP and via both the hypothalamus and the nucleus of solitary tract in the baroregulation, and that the intrinsic opioid systems are involved in the action of NPFF.

Hypothalamic dysfunction in Parkinson's disease patients.

Ten patients with idiopathic Parkinson's disease (PD) (3 men and 7 women, group A) who had received no treatment for the disease; 102 patients with PD (36 men and 66 women, group B) who had undergone treatment and 45 healthy volunteers (15 men and 30 women, control group) were subject to thyrotropin-releasing hormone (TRH) tests and levodopa tests. In group A basal plasma prolactin (PRL) levels were significantly higher than in the controls both before and during treatment. Peak plasma PRL levels during TRH tests were significantly higher before treatment, but returned to the control levels during treatment. Nadir plasma PRL levels during levodopa tests were significantly increased before and during treatment. In group B basal plasma thyroid-stimulating hormone (TSH) and PRL levels were significantly higher than in the control group. Peak plasma PRL levels during TRH tests and nadir plasma PRL levels during levodopa tests were also significantly increased. The results strongly suggest a disturbance of pituitary hormone secretion due to hypothalamic dysfunction in PD patients.