Vladimír Štrbák

Maturation of the Pituitary-Adrenal Function in Rat Fetuses

Plasma adrenocorticotropic hormone (ACTH) and corticosterone were detectable in fetal plasma on day 16 of pregnancy. Thereafter, the levels of both hormones increased steadily in a parallel manner and reached a peak on day 19 of pregnancy. Administration of an antiserum anti-rat corticotropin-releasing factor (CRF) to pregnant rats was followed by a significant decrease in fetal plasma corticosterone as early as day 17. Plasma ACTH measured under the same experimental conditions on day 19 of gestation was also significantly decreased. Similar results have been obtained with fetal plasma collected from adrenalectomized pregnant rats, indicating that the plasma corticosterone decrease in fetuses after immunoneutralization of CRF reflects changes in fetal adrenal secretion and not a diminution of corticosterone transfer from the maternal to the fetal circulation. These results show that endogenous CRF begins to play a physiological role in the regulation of ACTH and corticosterone secretion as early as in 17-day-old fetuses. This effect may occur before the connections between the neurosecretory CRF axons and the hypophysial portal capillaries have been established. Therefore, endogenous CRF may enter the hypophysial portal circulation after intercellular diffusion in hypothalamic tissue.

Four-Week Ethanol Intake Decreases Food Intake and Body Weight But Does Not Affect Plasma Leptin, Corticosterone, and Insulin Levels in Pubertal Rats

Long-term intake of ethanol decreases food intake and inhibits growth in experimental rats. The aim of this study was to determine the effect of 4-week oral ethanol ingestion on plasma leptin and adrenal function. Male 45-day-old Wistar rats were divided into three groups: absolute control (AC), ethanol (E) administered 10% (wt/vol) ethanol instead of tap water, and pair-fed (PF) given an amount of food corresponding to the food intake of E animals. E rats consumed less pelleted diet (74% cumulative total intake); however, this caloric deficit was compensated by ethanol ingestion. Net water intake in E animals was 76% of that in the control groups. The body growth of both E and PF rats was stunted compared with AC animals, but E rats were heavier than PF rats. The plasma leptin level was similar in E and AC and decreased in PF animals. There were no differences in plasma osmolality or glycemia among the three groups. Plasma insulin was decreased in PF compared with both AC and E rats. Plasma corticosterone was not affected by ethanol, but was increased in the food-restricted (PF) group. Although there were no differences in basal adrenal corticosterone production in vitro, there was a slightly higher response to corticotropin (ACTH) in E rats. We conclude that drinking 10% ethanol decreased the dietary intake and body growth. These changes were not mediated by plasma leptin changes. Although alcohol ingestion and its energy content theoretically normalized the total energy intake and prevented the decrease of plasma leptin, the growth of young rats was inhibited. Drinking 10% ethanol instead of tap water for 4 weeks did not stimulate basal adrenal activity.

Resveratrol Inhibits Electrical Activity and Insulin Release from Insulinoma Cells by Block of Voltage-Gated Ca2+ Channels and Swelling-Dependent Cl- Currents

The phytostilbene resveratrol (RV) improves the metabolic state in animal models by increasing the insulin responsiveness of tissues and there is evidence that RV affects insulin secretion from native β-cells and insulinoma cells. In whole cell patch clamp experiments on clonal rat INS-1E cells we used high extracellular glucose (20 mM), extracellular hypotonicity (30%) or tolbutamide (100 μM) to elicit membrane depolarizations and electrical activity. Application of RV (50 μM) repolarized the cells, terminated electrical activity and prevented the hypotonicity-induced depolarization. These effects were fully reversible and intermittent application of RV restored tolbutamide-induced electrical activity after desensitization. Glucose-induced depolarization was counteracted by RV in presence of iberiotoxin (50 nM), showing that the RV effect does not depend on BKCa channel activation. RV dose-dependently inhibited KATP currents, L- and T-type Ca2+ currents and swelling-dependent Cl- currents evoked by either hypotonicity or high extracellular glucose - ion conductances crucially involved in regulating the electrical activity of insulin secreting cells. We further show that RV blunts glucose-induced, but not basal insulin release. Our results indicate that RV counteracts/prevents stimulus-induced cell membrane depolarization and electrical activity by blocking voltage-gated Ca2+- and swelling-dependent Cl- currents despite the inhibition of KATP currents.

HYPOSMOLAR MEDIUM AND ETHANOL IN ISOSMOTIC SOLUTION INDUCE THE RELEASE OF THYROTROPIN-RELEASING HORMONE (TRH) BY ISOLATED RAT PANCREATIC ISLETS

Cell swelling induced by hypotonic medium or small isotonic permeant molecules results in an immediate secretory response in various types of cells. We have expanded exploration of this phenomenon by examining the effect of either isotonic ethanol or hyposmotic medium on the release of TRH by freshly isolated islets of Langerhans in static incubation and perifusion. Ethanol (40, 80 or 160 mM in isotonic solution) dose-dependently evoked the release of TRH by statically incubated islets. The dynamics of TRH release induced by 80 mM isotonic ethanol or 30% hypotonic medium were similar to those induced by 50 mM KCl, with the highest secretion rate during the first 5 min of incubation irrespective of the duration of stimulation. Ca2+ depletion of the incubation medium abolished the response to 50 mM KCl but did not diminish the response to 80 mM isotonic ethanol. We conclude that osmotic stimuli known to induce cell swelling also induce release of TRH by isolated pancreatic islets.

Cell volume and peptide hormone secretion.

In general way cell swelling evokes and shrinking inhibits exocytosis of proteins and peptides stored in secretory vesicles from various types of cells. Dynamics of this type of hormone secretion is indistinguishable from that induced by specific secretagogue. Peculiarities of swelling-induced secretion indicate an involvement of the unique signaling pathway. Hyposmotic stimulation of insulin secretion is independent from the extra- and intracellular Ca(2+), does not involve other intracellular mediators of glucose stimulation, and could not be inhibited by noradrenaline. Swelling-induced peptide secretion is not essential for cell volume control. Hyposmotic stimulation is a useful research tool when natural or pharmacological secretagogue is unknown: Thyrotropin releasing hormone release from the heart slices, pancreatic islets and various brain structures was characterized by the stimulation by hypotonic medium. Swelling-induced exocytosis possesses limited selectivity; cells involved in water and salt regulation retain their specific response to osmotic stimuli; hypotonic medium evokes thyrotropin releasing hormone but not oxytocin (OT) release from hypothalamic paraventricular nucleus. Specific response (release after hyperosmotic stimulation) of intranuclear OT secretion in the paraventricular nucleus and the supraoptic nucleus could be obviated by GdCl(3) and at these conditions OT release to swelling-inducing stimuli emerged. Swelling-induced hormone secretion can have pathophysiological implications. For example, a shift to anaerobic glycolysis and production of metabolites occurring in ischemia results in the increased intracellular osmolarity and cell swelling. Peptides and proteins released after swelling could play an important role in the pathophysiology of ischemia and be mediators of local or remote preconditioning when factors released at the place of ischemia have protective effect against ischemia-reperfusion injury. Moreover, the ischemic disruption of the osmotic receptors could result in a syndrome of inappropriate hormone secretion.

Intracerebroventricular oxytocin administration in rats enhances object recognition and increases expression of neurotrophins, microtubule-associated protein 2, and synapsin I

Brain oxytocin regulates a variety of social and affiliative behaviors and affects also learning and memory. However, mechanisms of its action at the level of neuronal circuits are not fully understood. The present study tests the hypothesis that molecular factors required for memory formation and synaptic plasticity, including brain‐derived neurotrophic factor, neural growth factor, nestin, microtubule‐associated protein 2 (MAP2), and synapsin I, are enhanced by central administration of oxytocin. We also investigated whether oxytocin enhances object recognition and acts as anxiolytic agent. Therefore, male Wistar rats were infused continuously with oxytocin (20 ng/µl) via an osmotic minipump into the lateral cerebral ventricle for 7 days; controls were infused with vehicle. The object recognition test, open field test, and elevated plus maze test were performed on the sixth, seventh, and eighth days from starting the infusion. No significant effects of oxytocin on anxious‐like behavior were observed. The object recognition test showed that oxytocin‐treated rats significantly preferred unknown objects. Oxytocin treatment significantly increased gene expression and protein levels of neurotrophins, MAP2, and synapsin I in the hippocampus. No changes were observed in nestin expression. Our results provide the first direct evidence implicating oxytocin as a regulator of brain plasticity at the level of changes of neuronal growth factors, cytoskeletal proteins, and behavior. The data support assumption that oxytocin is important for short‐term hippocampus‐dependent memory.

Cell volume induced hormone secretion: studies on signal transduction and specificity.

Cell swelling causes an immediate secretory response in various cell types. Induced secretion possesses some unique features suggesting the involvement of a specific signal transduction pathway. The effect of 10-20 µM GdCl3, 100 µM HgCl2, 1-100 µM indomethacin and 1-20 µM nordihydroguaiaretic acid (NDGA) on cell swelling-induced hormone secretion (isosmotic 80 mM ethanol or 15-30% hyposmotic medium) from incubated rat hypothalamic paraventricular nucleus (PVN) and posterior pituitary (oxytocin and TRH), isolated pancreatic islets (TRH) and perifused anterior pituitary cells (prolactin) were examined. To determine how general the effect of cell swelling is on exocytotic secretion, the release of two different neurohormones (thyrotropin releasing hormone -TRH and oxytocin) from the same tissue explant were studied. Both hyposmotic medium or isosmotic ethanol containing medium induced immediate TRH and prolactin release from the tested tissues.The effect of GdCl3, HgCl2, NDGA or indomethacin showed no inhibition of cell swelling induced secretion. In contrast to TRH, oxytocin release was not induced by isosmotic ethanol containing medium from the PVN or posterior pituitary. CONCLUSION: These data indicate that signal transduction leading to exocytosis after cell swelling does not involve GdCl3 sensitive stretch activated channels, mercury sensitive aquaporins, or indomethacin and NDGA sensitive mediators including prostaglandins and leukotriens. Cell swelling-induced exocytosis possesses limited selectivity; cells specifically involved in water and salt regulation retain their specific response to osmotic stimuli.

Four-week ethanol drinking increases both thyrotropin-releasing hormone (TRH) release and content in rat pancreatic islets.

Ethanol exerts profound effects on the endocrine and exocrine pancreas. Some effects of chronic alcohol consumption on insulin secretion in response to glucose load are similar to those of TRH gene disruption. TRH is present in insulin-producing B-cells of the islets of Langerhans; its role in this location is still not fully explored. To examine the possible effect of long-term in vivo ethanol treatment on pancreatic TRH we compared three groups of rats: a 10% (wt:vol) ethanol-drinking group (E), absolute controls (AC) and pair-fed (PF) group with solid food intake corresponding to that of E. The fluidity of pancreatic membranes was not affected by chronic in vivo exposure of rats to ethanol, but was significantly decreased in PF group. Four-week treatment resulted in significantly higher TRH content in isolated islets of the E group and increased basal and 80 mM isotonic ethanol-induced secretion compared to AC and PF. Plasma levels of insulin, C-peptide, IGF-I, and glycemia were, however, not affected by ethanol treatment. Cell swelling, which can be induced by the presence of permeants (e.g. ethanol) in an isotonic extracellular medium, is a strong stimulus for secretion in various types of cells. In the present study, isosmotic ethanol (40, 80, and 160 mM) induced dose-dependent release of TRH and insulin from adult rat pancreatic islets in vitro. The same concentrations were not effective when applied in a hyperosmotic medium (addition of ethanol directly to the medium), thus indicating the participation of cell swelling in the ethanol-induced secretion. In conclusion, chronic ethanol treatment significantly affected pancreatic TRH and this effect might be mediated by cell swelling. The role of these changes in the profound effect of ethanol on the endocrine and exocrine pancreas remains to be established.

Long-term action of potassium bromide on the rat thyroid gland

Male rats fed by a standard diet with determined of bromine and iodine content were exposed to a 133-day oral administration of KBr (100, 200, 400 mg Br-/l drinking water). Their thyroid glands showed increased growth of the epithelial cells reflected by a microfollicular rearrangement of the parenchyma due to proliferation of very small follicles with a low or zero content of colloid. Morphometric analysis of thyroids of Br(-)-exposed animals revealed a significant decrease in the volume of intrafollicular colloid and marked increase in the number of the smallest follicles (areas up to 100 and 100-300 micron 2). In addition, the nuclei of thyrocytes showed an increased number of mitoses. The vascularization was increased as well. In the blood plasma of the Br(-)-exposed animals the T4 concentration was significantly decreased in dependence on the bromine concentrations. Thyroglobulin immunoreactivity in the colloid of Br(-)-exposed animals decreased after administration of 400 mg Br-/l drinking water. Increasing concentrations of Br- in the drinking water caused an increased bromine concentration in the thyroid, a decreased iodine content and a decreased I/Br molar ratio. The changes in the rat thyroid caused by long-term administration of 100 mg Br-/l were similar to hyperplastic parenchymal goitre and were comparable to those induced in previous experiments by the same bromine concentration administered over a 16- and 66-day period respectively.

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.