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.

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.

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.

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.

Hyperosmotic environment blunts effectivity of ischemic preconditioning against ischemia-reperfusion injury and improves ischemic tolerance in non-preconditioned isolated rat hearts.

Several studies have shown that diabetes mellitus modulates heart resistance to ischemia and abrogates effectivity of cardioprotective interventions, such as ischemic preconditioning (IP). The aim of this study was to evaluate whether the effect of hyperglycemic conditions on the severity of ischemia-reperfusion (I/R) injury in preconditioned and non-preconditioned hearts (controls, C) is related to changes in osmotic activity of glucose. Experiments were performed in isolated rat hearts perfused according to Langendorff exposed to 30-min coronary occlusion/120-min reperfusion. IP was induced by two cycles of 5-min coronary occlusion/5-min reperfusion, prior to the long-term I/R. Hyperosmotic (HO) state induced by an addition of mannitol (11 mmol/l) to a standard Krebs-Henseleit perfusion medium significantly decreased the size of infarction and also suppressed a release of heart fatty acid binding protein (h-FABP - biomarker of cell injury) from the non-IP hearts nearly to 50 %, in comparison with normoosmotic (NO) mannitol-free perfusion. However, IP in HO conditions significantly increased the size of infarction and tended to elevate the release of h-FABP to the effluent from the heart. The results indicate that HO environment plays a cardioprotective role in the ischemic myocardium. On the other hand, increased osmolarity, similar to that in the hyperglycemic conditions, may play a pivotal role in a failure of IP to induce cardioprotection in the diabetic myocardium.

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.

Acute effects of hypothalamic ablation on plasma thyrotropin and prolactin concentrations in the suckling rat: evidence that early postnatal pituitary-thyroid regulation is independent of hypothalamic control.

Hypothalamic ablation was performed at various periods postnatally in animals previously administered propylthiouracil to raise plasma TSH concentrations. There was no significant change in plasma Tsh up to 8 h after hypothalamic ablation in pups 1--4 days old, whereas hypophysectomy of such pups produced a 60% fall in plasma TSH within 4 h. By the 5th postnatal day, hypothalamic ablation produced a 30% fall in plasma TSH within 4 h (P less than 0.05). By the 12th postnatal day and thereafter, the fall in plasma TSH after hypothalamic ablation was not significantly different from that seen in adults, except in 30-day-old rats in which there was a lesser effect of hypothalamic ablation on plasma TSH (P less than 0.01 in comparison to 23-day-old and adult groups). The greatest effect of hypothalamic ablation on plasma TSH was in 45-day-old animals (P less than 0.01 in comparison to adults). No significant change was produced in plasma PRL within 4 h postoperatively at any age. Our data indicate that regulation of TSH secretion in the rat is independent of hypothalamic control until after the 5th postnatal day and is fully developed by day 12. This corresponds temporally with the postnatal rise of plasma TSH, T4, and T3 and hypothalamic TRH to adult concentrations and indicates maturation of the hypothalamic regulation of TSH secretion.

Thyrotropin secretory response to thyrotropin-releasing hormone in the hypothyroid perinatal rat: further evidence of thyrotroph independence of the hypothalamus during early ontogenesis.

Propylthiouracil fed to pregnant rats for the last week of gestation to induce maternal and fetal hypothyroidism induced a 3-fold rise in plasma TSH concentration in the newborn pups compared to a 4-fold rise in their mothers. Subcutaneous administration of 1 ng/g BW TRH caused a greater rise in plasma TSH in the hypothyroid pups than in their mothers. These results, in combination with published data, indicate that the apparent independence of pituitary-thyroid function from TRH control during early ontogenesis in the rat is primarily due to delayed maturation of the hypothalamic TRH system.

Changes of adrenal catecholamines and their synthesizing enzymes during ontogenesis and aging in rats.

Male Wistar specific-pathogen-free rats aged 2, 7, 17, 30, 60, 120, 200, 360 and 600 days, all killed in experiment on the same day, were examined. The body weight significantly increased until the 200th day, the weight of adrenals until the 120th day and the adrenal protein content until the 30th day of life. The adrenaline content of the adrenals increased continuously during the 600 days studied. Adrenal noradrenaline content increased rapidly over the first 17 days, remained at a stable level until the 120th day, and rose to a higher level after 200 days. The activity of adrenal catecholamine-synthesizing enzymes also increased with age: tyrosine hydroxylase gradually increased until the 360th day, dopamine-beta-hydroxylase and phenylethanolamine-N-methyl transferase until the 200th day. Our results demonstrate that, in the rat, during development there is a gradual increase of adrenal weight, adrenaline content, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase activity until maturation (120th day), whereas the adrenal noradrenaline content reaches the adult values earlier, around the 17th day. During aging, adrenal catecholamines significantly increase when compared to young-adult rats (120-day-old), probably due to the elevated activity of the adrenal catecholamine-synthesizing enzymes. The increased adrenal catecholamine levels in old animals might be connected with a higher incidence of cardiovascular diseases in aged.

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.

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.

Glucose stimulates and insulin inhibits release of pancreatic TRH in vitro.

OBJECTIVE: Pancreatic TRH is present in insulin-producing B-cells of the islets of Langerhans. There is fragmentary evidence that it may be involved in glucoregulation. The aim of our present study was to analyze how glucose and insulin affect TRH secretion by the pancreatic islets. DESIGN: Isolated pancreatic islets were incubated with different concentrations of glucose, insulin and glucagon, and TRH release was measured. RESULTS: In the present study, 6 and 12mmol/l d-glucose caused significant TRH release from isolated adult rat pancreatic islets when compared with that in the presence of the same concentrations of biologically ineffective l-glucose. Thirtymmol/l d-glucose was also ineffective, but this was not due to depression of secretion by hyperosmolarity since isosmotic compensation for the high glucose addition did not restore its stimulatory effect. Five micromol/l dibutyryl cyclic 3',5'-adenosine monophosphate (db-cAMP) increased both basal and glucose-stimulated TRH release, but this effect was not seen with 50micromol/l db-cAMP. Stimulation of phosphodiesterase by imidazole resulted in decreased basal but not glucose-stimulated release of TRH. Glucagon (10(-7)mol/l) did not affect either basal or glucose-stimulated release of TRH, while insulin (10(-7) and 10(-6)mol/l) inhibited both. CONCLUSION: Our present data showing that glucose stimulates and insulin inhibits pancreatic TRH release are compatible with the possibility that this substance may play a role in glucoregulation.

Body mass, plasma leptin, glucose, insulin and C-peptide in offspring of diabetic and non-diabetic mothers.

OBJECTIVE: The aim was to investigate the relationship between body mass index (BMI), plasma leptin, glucose, insulin and C-peptide levels in the offspring of diabetic mothers (DM) and non-diabetic healthy mothers (HM). DESIGN: Seventy-two offspring (37 girls and 35 boys, age 4-20 years) of DM were investigated in a prospective study. Those 14-16 years old (Tanner stage II-IV) were compared with age-matched offspring of HM (33 girls and 33 boys). RESULTS: BMI strongly correlated with plasma leptin concentration in the offspring of both DM and HM children. There were higher BMI and plasma leptin and glucose levels in DM than in HM children. There was no difference in plasma insulin or C-peptide levels between HM and age-matched DM children. There was a highly significant positive correlation between plasma leptin and C-peptide in boys of DM. CONCLUSIONS: The higher plasma leptin found in the offspring of DM reflects their higher BMI. A moderately high but still normal glycemia might be a preclinical sign of insulin resistance or other disturbance of glucoregulation.

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.

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.

Effects of sauna and glucose intake on TSH and thyroid hormone levels in plasma of euthyroid subjects.

The effect of sauna on thyroid function parameters and its modification by glucose was studied in young euthyroid male volunteers. A 30-minute stay in sauna resulted in an increase in plasma TSH; the response was exaggerated if glycemia had been increased by oral glucose intake at the beginning of the experiment. Plasma rT3 also increased in sauna, this response was, however, blunted by the higher glycemia. TSH response to sauna was definitely present in young men (aged 20 to 25) and absent in middle-aged ones (50 to 55). To explore the mechanism of the effect of increased glycemia, TRH tests were performed and dopamine infusions were administered with and without glucose pretreatment. Increased glycemia did not affect TSH and T3 response to TRH in young volunteers; however, 90 minutes after the administration, plasma rT3 levels were significantly lower in glucose pretreated subjects than in those receiving TRH injections after water pretreatment. Simultaneous infusion of glucose prevented the inhibitory effect of dopamine infusion on plasma TSH. It was concluded that glucose directly modulates the effect of sauna on plasma TSH at a suprapituitary level, while the inhibiting effect of glucose on plasma rT3 response to sauna and TRH is probably mediated by the insulin effect on thyroid hormone metabolism.

Different regulation of thyrotropin releasing hormone content and release in paraventricular nucleus (PVN) and median eminence (ME) of rat hypothalamus during in vitro incubation.

TRH is present throughout the central nervous system possessing many different functions. Only TRH synthesized in the hypothalamic PVN and transported to the ME regulates anterior pituitary secretion of thyrotropin. To investigate this hypophysiotropic system, we have developed a method using dissected rat PVN and ME, representing mostly cell bodies and nerve terminals respectively, of the same TRH system. Tissues were incubated for four 30 min periods each in Locke's medium with alternatively normal and high KCl concentration. Repeated KCl-induced membrane depolarization resulted in significantly increased TRH release from both tissues (15% of TRH content) which was dependent on Ca2+ influx. Some important differences were found in the regulation of TRH in the PVN and ME: Comparison of TRH content in the tissue before and after incubation with the amount of secreted TRH showed new production of TRH during incubation in the ME, but not in the PVN. Frequent medium replacement during depolarization revealed that TRH secretion at the level of the ME, but not the PVN, is probably inhibited by some substance released during incubation. These data suggest that there is a different regulation of TRH in the PVN and ME and that TRH secreted by the isolated PVN may come mostly from the perikarya and represent paracrine neurohormone secretion.

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.

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.