Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Comparison of pancreatic and hypophysiotropic TRH systems.

The thyrotropin-releasing hormone (TRH) is a molecule with widespread distribution through many organ systems. The function of TRH is probably not identical in each system so that TRH synthesis and secretion may be unique for each system under specific experimental conditions. The present study was designed to explore the common and diverse features of the regulation of TRH encoded with the same gene in two different organs: hypophysiotropic hypothalamus and pancreatic islets. During in vitro incubation, the TRH content in hypothalamic structures remained stable while that in isolated pancreatic islets increased sharply. In contrast to the pancreatic islets, exposure to different concentrations of D-glucose did not affect TRH release from the hypothalamic paraventricular nucleus or median eminence. This divergence in the regulation of the hypophysiotropic and pancreatic TRH systems may be related to differences in the role of TRH produced in these tissues.

Hypo-osmolarity stimulates and high sodium concentration inhibits thyrotropin-releasing hormone secretion from rat hypothalamus.

The hypothalamic paraventricular nucleus, representing cell bodies in which thyrotropin-releasing hormone is synthesized, and the median eminence, representing nerve terminals, were incubated in vitro. Various hypo- and hyperosmotic solutions were tested to determine osmotic sensitivity of thyrotropin-releasing hormone secretion. High KCl (56 mM) causing membrane depolarization was used as a non-specific control stimulus to induce thyrotropin-releasing hormone secretion. A 30% decrease of medium osmolarity (from 288 to 202 mOsmol/l) increased thyrotropin-releasing hormone secretion from both the paraventricular nucleus and median eminence. A 30% decrease of medium NaCl content by its replacement with choline chloride did not affect basal thyrotropin-releasing hormone secretion. Increasing medium osmolarity with biologically inactive L-glucose did not affect basal or KCl-induced thyrotropin-releasing hormone secretion from either structure. Medium made hyperosmotic (350-450 mOsmol/l) by increasing the NaCl concentration resulted in a dose-dependent decrease of basal thyrotropin-releasing hormone secretion and abolished KCl-induced thyrotropin-releasing hormone secretion. If an osmotically equivalent amount of choline chloride was substituted for NaCl, there was no effect on thyrotropin-releasing hormone secretion, indicating a specific action of Na+. This study indicates a specific sensitivity to high concentrations of Na+ ions of both thyrotropin-releasing hormone-producing parvocellular paraventricular neurons and thyrotropin-releasing hormone-containing nerve terminals in the median eminence.

Chronic ethanol drinking and food deprivation affect rat hypothalamic-pituitary-thyroid axis and TRH in septum.

Because chronic ethanol ingestion may perturb thyroid function, we evaluated the effect of 4-wk of oral 10% ethanol ingestion on the hypothalamic-pituitary-thyroid (HPT) axis and septal thyrotropin-releasing hormone (TRH) in 200-g male Wistar rats. Animals were divided into three groups: absolute control receiving tap water and food ad libitum; ethanol group receiving food ad libitum and 10% ethanol as the sole source of drinking fluid; pair-fed group receiving tap water and an amount of food corresponding to the consumption of ethanol group. After 4-wk of treatment, the body weight of the ethanol group was 7% and of the pair-fed rats 19% lower than that of the absolute controls. Both chronic ethanol treatment and food deprivation produced a decrease in plasma thyroid-stimulating hormone (TSH). Pair-fed rats also had a lower plasma T3. Type I iodothyronine 5'-deiodinase activity in the liver was increased in the pair-fed and even more in the ethanol-treated group. The content and secretion in vitro of TRH from the hypothalamic paraventricular nucleus and median eminence were unchanged. TRH content in the septum was increased in both the ethanol and pair-fed groups. TRH secretion from the septum in vitro was lower in the pair-fed, but unchanged in the ethanol group. These data suggest that 4-wk of peroral ethanol intake affects thyroid function mostly at the extrahypothalamic level and that there is a contribution of concomitant food deprivation. Both ethanol treatment and food deprivation increased TRH content in the septum.

Both iso- and hyperosmotic ethanol stimulate release of hypothalamic thyrotropin-releasing hormone despite opposite effect on neuron volume.

Previous studies have indicated that isosmolar, but not hyperosmolar, ethanol induces in vitro gonadotropin-releasing hormone secretion from the basal hypothalamus, presumably by causing cell swelling. Moreover, ethanol reduces secretion of another hypothalamic neuropeptide vasopressin. We have studied the acute effect of ethanol on specific hypophysiotropic basal and K+-stimulated thyrotropin-releasing hormone secretion in vitro especially in relation to cell swelling. Isosmotic 40-160 mM ethanol increased thyrotropin-releasing hormone release from the hypothalamic paraventricular nucleus and median eminence in a dose-dependent manner. Both a 30% decrease of osmolarity and isosmotic 80 mM ethanol induced 12% swelling of hypothalamic neurons. Hyperosmotic 80 mM or 160 mM ethanol induced release of thyrotropin-releasing hormone from both hypothalamic structures but did not cause cell swelling (80 mM) or even induced cell shrinkage (160 mM). Depletion of medium Ca2+ did not affect thyrotropin-releasing hormone secretion caused by either isosmotic or hyperosmotic ethanol. Our data indicate that both iso- and hyperosmotic ethanol stimulated release of hypophysiotropic thyrotropin-releasing hormone despite opposite effects on neuron volume. The mechanism of ethanol action appears complex and variable depending on the type of cell and neuropeptide affected.