Oxytocin action on components of endoplasmic reticulum in hippocampal neuronal cells.

Despite the known importance of the endoplasmic reticulum (ER) in protein synthesis and vesicular transport, it is not clear whether neuropeptide and neuromodulator oxytocin can directly affect components of the ER in neuronal cells. Therefore, in the present study, we hypothesize that incubation of hippocampal neuronal cells in a presence of oxytocin 1) plays a role in the regulation of the expression of selected ER chaperone components and molecules involved in unfolded protein response pathway 2) affects distribution of the intracellular fluorescence signal highly selective for the ER. We found that oxytocin (1 µM) after 60 min significantly decreased the gene expression of oxidoreductase Ero1ß, chaperone glucose-regulated proteins (Grp) 78 and Grp94. A significant decrease in GRP78 protein levels in response to oxytocin treatment occurred after 30, 60 and 120 min. We also observed a time-dependent increase in calreticulin protein levels with a statistically significant increase observed after 360 min. We found that the dynamics of the ER network changes significantly within 2 h of incubation under the influence of oxytocin. In conclusion we have shown that ER chaperones, oxidoreductases and trafficking molecules in neuronal cells are changing in response to oxytocin treatment in a short-term scenario potentially relevant for growth of dendrites and axons.

Expression of synaptic proteins in the hippocampus is modulated by neonatal oxytocin treatment.

An alteration of oxytocin signaling during postnatal maturation of the brain could be associated with etiology of neurodevelopmental disorders among them autism. The aim of the present study was to examine the role of oxytocin in the regulation of expression of selected cell-adhesion molecules and scaffolding proteins in the hippocampus in early rat development. Oxytocin treatment (1 mg/ml, i.p., 50 µl/pup) at postnatal days P2-P3 resulted in the reduction of Neuroligin 3 gene expression, and was accompanied by lower SHANK1 and SHANK3 mRNA levels in the hippocampus at P5 day. Immunostaining revealed a clear trend for the lower density of Neuroligin 3 positive cells in the hippocampus and this trend has been significant in the CA3 hippocampal area. The significantly lower Neurexin 2ß mRNA levels were observed in response to oxytocin treatment, with no effect seen in the Neurexin 2α gene expression. No change has been observed in the gene expression of Neuroligin 1 and Neuroligin 2. Oxytocin induced an increase in the mRNA levels of Neuron-Specific Enolase (NSE) and a decrease in the mRNA levels of glial fibrillary acid protein (GFAP) - marker of astrocytes. Incubation of primary neuronal cells with oxytocin (1 µM, 48 h) stimulated a proliferation of NSE-positive cells. These results suggest that synaptic proteins could be under control of oxytocin in early stages of brain development. The changes of cell-adhesion molecule and scaffolding protein levels might be linked to the modulation of number of neuronal cells.

Activation of the Oxytocin Receptor Modulates the Expression of Synaptic Adhesion Molecules in a Cell-Specific Manner.

Synaptic cell adhesion molecules, including neurexins and neuroligins, mediate the formation and maintenance of connections between neuronal cells. Although neurexins and neuroligins are known to interact with each other in a calcium-dependent manner and several neuropeptides have been shown to act through G protein-coupled receptors to increase intracellular calcium levels, no studies have examined the role of the neuropeptide oxytocin in association with adhesion molecules. Given that oxytocin receptors are located on presynaptic and postsynaptic membranes and that oxytocin exerts direct effects on neuronal excitability, it could be hypothesized that oxytocin affects the expression of cell surface adhesion molecules. In the present study, we show that incubation in the presence of oxytocin (1 µM, 48 h) exerted cell-specific effects on the levels of neurexin 2α, neurexin 2ß, and neuroligin 3. Oxytocin significantly increased the mRNA expression levels of neurexin 2α, neurexin 2ß, and neuroligin 3 in SH-SY5Y, U-87MG, and primary cerebellar cells. The effect of inhibiting oxytocin receptors on the expression of neurexin 2ß was more dramatic in U-87MG cells than in SH-SY5Y cells. Oxytocin did not exert effects in primary corticohippocampal cells. Additionally, we measured the expression of selected GTPases to determine whether they could mediate the effects of oxytocin. Oxytocin induced a decrease in the mRNA level of Rac1 in U-87MG and primary cerebellar cells and exerted a stimulatory effect on the expression of RhoB at the gene and protein level in SH-SY5Y cells. These results suggest that the regulation of neurexins and neuroligins involves the activation of oxytocin receptors. These effects are likely mediated by the stimulation of RhoB GTPase, at least in certain types of cells.

Projection length stimulated by oxytocin is modulated by the inhibition of calcium signaling in U-87MG cells.

Neuropeptide oxytocin contributes to the regulation of glial cell morphology. The precise mechanisms, however, are not yet fully understood. In the present study, we have investigated whether an oxytocin-induced increase of intracellular calcium is required for cell extension in astrocyte-like U-87MG cells. Oxytocin (1 µM) significantly increased the length of the cell projections measured by the green-fluorescent protein labeled microtubule-associated protein after 48 h. The knockdown of oxytocin receptors (OXTR) in U-87MG cells prevented the elongation of the projections. Incubation of U-87MG cells in the presence of oxytocin, resulted in a significant increase of intracellular calcium, specifically blocked by the OXTR antagonist L-371,257. Both quercetin, which is a phosphoinositide 3-kinase inhibitor, and the phospholipase C inhibitor U-73122 reduced oxytocin-induced elevation of intracellular calcium concentration. Conversely, neither diltiazem, an L-type voltage-gated calcium channel blocker nor tetracaine, which is a blocker of the ryanodine receptors, showed an effect on intracellular calcium levels. Treatment of cells with quercetin, U-73122 and the voltage-gated calcium channel blockers cilnidipine, ω-agatoxin and mibefradil prevented the elongation of projections stimulated by oxytocin. Oxytocin treatment resulted in a significant increase in gene and protein expression of the scaffolding protein SHANK3. Our results clearly show that activation of OXTRs contributes to the elongation of cell projections in astrocyte-like U-87MG cells and that this effect is mediated by an extracellular calcium influx accompanied by an increase in scaffolding proteins expression.

Neurite Outgrowth Stimulated by Oxytocin Is Modulated by Inhibition of the Calcium Voltage-Gated Channels.

Neuropeptide oxytocin contributes to the regulation of the neuron differentiation and cell morphology. However, the precise mechanisms are not yet fully understood. Oxytocin receptor function and its coupling to calcium entry are obvious objects of interest in relation to the neuron morphology. Postsynaptic scaffolding proteins including SHANK proteins interact with other synaptic molecules and change dendritic morphology. SH-SY5Y neuroblastoma cell line represents a useful neurobiological in vitro model to study the short-term oxytocin effects on neurite outgrowth and underlying mechanisms. In the present study, we show that oxytocin induces an increase in the intracellular calcium in SH-SY5Y cells. Specificity of the calcium influx was verified by blockade of the oxytocin receptors with oxytocin receptor antagonist L-371,257. Neurite outgrowth stimulated by oxytocin was inhibited by specific voltage-gated calcium channel blockers. The exposure of SH-SY5Y cells to oxytocin resulted in a significant increase in the gene expression of SHANK1 and SHANK3 proteins. Overall, the present data indicate that oxytocin may contribute to the regulation of scaffolding proteins expression known to be associated with clusters of calcium channels at the cell membrane. It appears that oxytocin stimulated neurite outgrowth is, at least, in part dependent on the voltage-gated calcium channels.

Oxytocin Modulates Expression of Neuron and Glial Markers in the Rat Hippocampus.

Neuropeptides including oxytocin belong to the group of factors that may play a role in the control of neuronal cell survival, proliferation and differentiation. The aim of the present study was to investigate potential contribution of oxytocin to neuronal differentiation by measuring gene and protein expression of specific neuron and glial markers in the brain. Neonatal and adult oxytocin administration was used to reveal developmental and/or acute effects of oxytocin in Wistar rats. Gene and protein expression of neuron-specific enolase (NSE) in the hippocampus was increased in 21-day and 2-month old rats in response to neonatal oxytocin administration. Neonatal oxytocin treatment induced a significant increase of gene and protein expression of the marker of astrocytes - glial fibrillary acid protein (GFAP). Oxytocin treatment resulted in a decrease of oligodendrocyte marker mRNA - 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) - in 21-day and 2-month old rats, while no change of CD68 mRNA, marker of microglia, was observed. Central oxytocin administration in adult rats induced a significant increase of gene expression of NSE and CNPase. The present study provides the first data revealing the effect of oxytocin on the expression of neuron and glial markers in the brain. It may be suggested that the oxytocin system is involved in the regulation of development of neuronal precursor cells in the brain.

Downregulation of Oxytocin Receptor Decreases the Length of Projections Stimulated by Retinoic Acid in the U-87MG Cells.

Oxytocin is a neuropeptide widely expressed in the brain. Oxytocin plays a role in both proliferation and differentiation of various cells. Previous studies have suggested that oxytocin could affect the morphology of neuronal cells, therefore the objective of the present study was to test whether (1) oxytocin receptor stimulation/inhibition by specific ligands may change cell morphology and gene expression of selected cytoskeletal proteins (2) oxytocin receptor silencing/knockdown may decrease the length of cell projections (3) oxytocin receptor knockdown may affect human glioblastoma U-87MG cell survival. We confirmed the stimulatory effect of retinoic acid (10 µM) and oxytocin (1 µM) on projection growth. The combination of retinoic acid (10 µM) and oxytocin receptor antagonist (L-371,257, 1 µM) decreased projections length. Contrary to our assumptions, oxytocin receptor silencing did not prevent stimulation of length of projection by retinoic acid. Retinoic acid's and oxytocin's stimulation of projections length was significantly blunted in U-87MG cells with oxytocin receptor knockdown. Cell viability was significantly decreased in U-87MG cells with oxytocin receptor knockdown. Significantly higher levels of mRNA for cytoskeletal proteins drebrin and vimentin were observed in response to oxytocin incubation for 48 h. The data obtained in the present study clearly show that oxytocin induces formation and elongation of cell projections in astrocyte-like U-87MG cells. The effect is mediated by oxytocin receptors and it is accompanied by an increase in gene expression of drebrin and vimentin. Thus, oxytocin receptor signaling, particularly in the glial cells, may play an important role in native cell life, differentiation processes, and tumor progression, as well.

Mechanisms involved in the regulation of neuropeptide-mediated neurite outgrowth: a minireview.

The present knowledge, regarding the neuronal growth and neurite extension, includes neuropeptide action in the central nervous system. Research reports have brought much information about the multiple intracellular signaling pathways of neuropeptides. However, regardless of the differences in the local responses elicited by neuropeptides, there exist certain functional similarities in the effects of neuropeptides, mediated by their receptors. In the present review, data of the relevant studies, focused on G protein-coupled receptors activated by neuropeptides, are summarized. Particularly, receptors that activate phosphatidylinositol-calcium system and protein kinase C pathways, resulting in the reorganization of the neuronal cytoskeleton and changes in the neuronal morphology, are discussed. Based on our data received, we are showing that oxytocin increases the gene expression of GTPase cell division cycle protein 42 (Cdc42), implicated in many aspects of the neuronal growth and morphology. We are also paying a special attention to neurite extension and retraction in the context of neuropeptide regulation.

Acridin-3,6-dialkyldithiourea hydrochlorides as new photosensitizers for photodynamic therapy of mouse leukemia cells.

Acridin-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been evaluated as a new group of photosensitizers (PSs) for photodynamic antitumor therapy (PDT). Mouse leukemia cells L1210 were used for testing of AcrDTUs as the new PSs. The irradiation (UV-A light (365 nm), 1.05 J/cm(2)) increased cytotoxicity of all derivatives against L1210 cells more than ten times. The highest photocytotoxicity was found for propyl-AcrDTU with IC50=0.48±0.03 µM after 48 h incubation. A generation of the superoxide radical anion upon UV-A irradiation of propyl-AcrDTU was confirmed by in situ photochemical EPR experiments. To explain a mechanism of photocytotoxic action of AcrDTUs, an intracellular distribution of propyl-AcrDTU has been studied. It was found that AcrDTU in non-irradiated cells was not present in their nucleus but in the lysosomes and partly in the mitochondria, and sequestration of propyl-AcrDTU was dependent on pH in lysosomes. After irradiation, the cell death was induced by oxidative damage of lysosomal and mitochondrial membranes. Concerning the cell cycle, flow cytometry after PDT with propyl-AcrDTU showed a significant increase of the cells in the subG0 phase. Observed signs of necrosis, apoptosis, and autophagy indicate that PDT/AcrDTU leads to multiple cell death types (caspase independent apoptosis, necrosis, and autophagy).

Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of ß-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 µM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of ß-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells.

Intracellular distribution of 3,6-bis(3-alkylguanidino)acridines determines their cytotoxicity.

UNLABELLED: Cytotoxicity of two derivatives of 3,6-bis(3-alkylguanidino)acridines (GNDAs; pentyl- and hexyl-GNDA) was determined against three cell lines: a murine immortalized fibroblast cell line NIH-3T3, a human ovarian carcinoma cell line A2780, and a human neuroblastoma cell line SH-SY5Y. We found out that these GNDAs were cytotoxic against A2780 and NIH-3T3 cells but they showed only a marginal cytotoxicity against neuroblastoma cells SH-SY5Y. To explain differences in cytotoxicity, intracellular distribution of GNDAs was monitored. GNDAs were accumulated in A2780 and NIH-3T3 cells in the nuclei (fluorescence microscopy). In contrast to these cell lines, in SH-SY5Y cells, GNDAs were localized outside of the nuclei, at the plasma membrane and surroundings, extending also to the cytosol. This distribution of GNDAs was confirmed by an ImageStream Flow Cytometer. Acetylcholinesterase (AChE) activity in the SH-SY5Y cells decreased upon incubation with GNDAs. Kinetic studies showed that GNDAs were able to inhibit AChE by the same mode as tacrine (9-amino-1,2,3,4-tetrahydroacridine), a known inhibitor of AChE. A low cytotocity of GNDAs against SH-SY5Y cells could be caused by their affinity to AChE (the enzyme is localized mainly at the plasma membrane). The interaction of GNDAs with AChE may affect their intracellular distribution and consequently the cytotoxicity. KEYWORDS: acetylcholinesterase, acridine, neuroblastoma cell line SH-SY5Y.

Prolactin increases expression of cytoskeletal proteins in SK-N-SH cells.

Although many studies have demonstrated the role of prolactin in the central nervous system, there is a considerable lack of known effects of prolactin on the parameters of neurogenesis and neuronal differentiation. The aim of the present study was to test whether prolactin changes gene expression and protein levels of nestin and microtubule-associated protein 2 (MAP2) in neuroblastoma (SK-N-SH) and glioblastoma (U-87MG) cells. Nestin and MAP2 represent cytoskeletal proteins associated with neuronal differentiation and they contribute to radial growth of the axons, dendrites and glial processes. SK-N-SH and U-87MG cells were exposed to prolactin (10 nM) for 48 h. Total mRNA was extracted. After reverse transcription, qPCR with specific primers for nestin and MAP2 was performed. The levels of proteins were measured by the In-Cell Western assay. Mitochondrial activity test was used to evaluate the viability of cells under the influence of prolactin. Incubation with 10 nM prolactin did not change the viability, either in SK-N-SH or in U-87MG cells. Prolactin significantly increased the gene expression and protein levels of both nestin and MAP2 in SK-N-SH cells, while no significant changes were observed in U-87MG cells. The presented data suggest that prolactin is linked to the regulation of cytoskeletal proteins in the neuronal type of cells and might be important for their differentiation.

[Mechanisms of growth of neuronal axons and dendrites]. / Mechanizmy zmien dlzky axónov a dendritov neurónu.

Brain development is determined by neuronal differentiation including changes of cell polarity and asymetric growth of neuronal processes. Although, there are many unkown factors contributing to changes of lenght of neuronal cones, mounting experimental and review papers focus on changes of growth conus and role of axonal transport. In particular, mechanisms of actin/microtubule polymerisation and depolymerisation are important. Role of intracellular calcium is also significant. Normal and properly timed changes of lenght of axons and dendrites are dependent on interaction of neurons and glia. Moreover, regeneration of injured axons is dependent on growth factors secreted from glial cells. The aim of the present study is characterisation of the most important mechanisms underlying changes of lenght of neurites.

Effect of cellular cholesterol changes on insulin secretion by tumor cell lines.

UNLABELLED: Glucose and cell swelling induce insulin secretion by alternative signaling pathways. Swelling-induced secretion is in most systems independent of calcium and various mediators of glucose stimulation. Comparison of two insulinoma tumor cell lines revealed surprising difference; INS-1E cells in contrast to INS-1 cells and isolated rat pancreatic islets do not respond to hypotonicity in the presence of calcium. To delineate the role of cholesterol the effect of its extraction or addition on the insulin secretion in response to glucose and cell swelling was compared. INS-1E cells have significantly higher cholesterol content than INS-1 cells (58.5 ± 2.9 and 46.3 ± 2.5 mg chol/mg prot respectively). After cholesterol desorption by 1.0, 5.0 and 10.0 mM of carboxymethyl-ß-cyclodextrin, methyl-ß-cyclodextrin, or 2-hydroxypropyl-ß- cyclodextrin the response to hypotonicity in INS-1E cells emerged. On the contrary, supplementation of INS-1 cells with cholesterol inhibited their response to cell swelling. Cyclodextrin pretreatment inhibited glucose-induced insulin secretion from INS-1 cells while INS-1E cells were more resistant to their effect. CONCLUSION: Cellular cholesterol content substantially affects secretory process; both high and low levels could be inhibitory. Absence of swelling-induced insulin secretion in INS-1E cells despite adequate response to glucose is related to their high cholesterol content. Optimal cholesterol concentration is different for either type of stimulation; swelling-induced mechanism is more sensitive to higher cholesterol content. The difference is likely to reflect involvement of sequential type exocytosis after cell swelling. Sensitivity of secretory processes suggests that either hypercholesterolemia or excessive effort to decrease plasma cholesterol in patients could have adverse effect on insulin secretion.

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli.

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.

Thyrotropin-releasing hormone in rat heart: effect of swelling, angiotensin II and renin gene.

AIM: This study was performed to examine thyrotropin-releasing hormone (TRH) secretion and regulation in rat heart. METHODS: Expression of prepro-TRH gene in left atrium and left ventricle was studied by RT-PCR. TRH secretion from slices of left auricle and left ventricle in response to cell swelling (induced by hypotonic medium or ethanol in isosmotic medium), angiotensin II and losartan and their combinations was studied. RESULTS: RT-PCR revealed two times higher prepro-TRH expression in left auricle than left ventricle. In transgenic rats with extra copy of mouse renin gene a marked increase of prepro-TRH expression in the heart was noted but the relative difference between left atrium and left ventricle persisted. The swelling stimulated TRH release from both left auricle and left ventricle and this stimulation could not be inhibited by bumetanide. Angiotensin II (10 nmol L-1) added into medium significantly decreased basal secretion of TRH. The inhibiting effect of Angiotensin II was prevented by 1 micromol L-1 losartan, an angiotensin II AT1 receptor blocker. When angiotensin II and hypotonicity were applied simultaneously, swelling-induced secretion persisted. CONCLUSION: TRH secretion from heart slices has attributes of regulated secretion--depending on the stimulus it could be either stimulated or inhibited. Renin positively affects prepro-TRH expression in the heart. Angiotensin II inhibits TRH secretion from heart tissue by a mechanism involving AT1 receptors. Swelling-induced TRH secretion overrides inhibitory effect of angiotensin II. Swelling could be a useful tool when natural or pharmacological secretagogue is unknown. Peptides and proteins released by swelling could be mediators of local and remote ischaemic preconditioning protecting from subsequent ischaemia.

The effect of swelling on TRH and oxytocin secretion from hypothalamic structures.

1. Cell swelling induces exocytosis of material stored in secretory vesicles resulting in a secretory burst of peptidic hormones or enzymes from various types of cells including endocrine cells and neurons. We have previously shown that swelling-induced exocytosis possesses limited selectivity; hypotonic medium evokes TRH but not oxytocin release from hypothalamic paraventricular nucleus (PVN) and neurohypophysis (NH). 2. It is the aim of this study to ascertain whether the swelling-induced oxytocin secretion could be unmasked by the inhibition of specific osmotic response using Ca(2+)-free medium and GdCl(3), an inhibitor of stretch activated channels. 3. Oxytocin release from the PVN was stimulated by the hypotonic medium only in the presence of 50 or 100 microM GdCl(3.) Oxytocin release from supraoptic nucleus (SON) was also stimulated by the Ca(2+)-free hypotonic medium in the presence of GdCl(3). Oxytocin secretion from the NH was not stimulated even in the presence of GdCl(3), both in Ca(2+) containing and Ca(2+)-free medium. TRH response to swelling-inducing stimulus was not affected by the presence of GdCl(3). 4. An intranuclear oxytocin secretion to hyposmotic stimulation within the PVN and the SON could be unmasked by the inhibiting specific response by GdCl(3). At these conditions general secretory response to swelling-inducing stimuli emerged. Secretion of oxytocin from the NH was not affected by any of these treatments. 5. Peptides and proteins released after cell swelling can play an important role in the pathophysiology of ischemia and could be mediators of local or remote preconditioning. Disruption of mechanosensitive gating in magnocellular neurosecretory cells could result in an inadequate secretory response (e.g. stimulation instead of inhibition and vice versa) of hormones engaged in water and salt metabolism regulation.

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats.

Neonatal STZ (nSTZ) treatment results in damage of pancreatic B-cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secretion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 microg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro. The expression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glucose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.

Effect of immobilization on in vitro thyrotropin-releasing hormone release from brain septum in wild-type and corticotropin-releasing hormone knock-out mice.

There is considerable evidence linking alcohol consumption, sedation, and thyrotropin-releasing hormone (TRH) in the brain septum. We have shown that ethanol in clinically relevant concentrations can in vitro induce TRH release from the septum by a mechanism involving neuronal swelling. Corticotropin-releasing hormone-deficient (CRH-KO) mice serve as an interesting model to help us understand the role of CRH in the regulation of different neuroendocrine systems. The aim of this study was to compare TRH release activity in the brain septum at basal and stress conditions in CRH-KO mice and their wild-type (WT) littermates. Experimental mice were decapitated immediately or 3 h after single (2 h) or repeated (seven times for 2 h daily) immobilization stress. The brain septum was immediately cut out and incubated to measure basal-, ethanol-, and hyposmosis-stimulated TRH release in vitro. Ethanol in isosmotic medium or hyposmotic medium stimulated TRH release from mice septal explants from WT and CRH-KO mice. The response was disturbed immediately after immobilization and recovered 3 h later. Our results show that immobilization stress transiently affects the TRH system in brain septum. Inborn absence of CRH does not affect septal TRH and its response to ethanol before and 3 h after immobilization.

Antioxidant properties of carboxymethyl glucan: comparative analysis.

Antioxidative capabilities of carboxymethylated (1 --> 3)-beta-D-glucan from Saccharomyces cerevisiae cell wall, alpha-tocopherol, and mannitol against lipid peroxidation in phosphatidylcholine liposomes induced by OH. radicals produced with Fenton's reagent (H(2)O(2)/Fe(2+)) were studied using absorption ultraviolet-visible spectrophotometry. It was found that (1 --> 3)-beta-D-glucan is an antioxidant with the scavenging ability lying between that of alpha-tocopherol, which is known to be incorporated in the lipid bilayer, and the water-soluble antioxidant, mannitol.