Ghrelin-Induced Enhancement of Vasopressin and Oxytocin Secretion in Rat Neurohypophyseal Cell Cultures.

The effects of ghrelin on vasopressin and oxytocin secretion were studied in 13-14-day cell cultures of isolated rat neurohypophyseal tissue. The vasopressin and oxytocin contents of the supernatant were determined by radioimmunoassay after a 1- or 2-h incubation. Significantly increased levels of vasopressin and oxytocin production were detected in the cell culture media following ghrelin administration, depending on the ghrelin doses. The oxytocin level proved to be more elevated than that of vasopressin. The increase of vasopressin and oxytocin secretion could be totally blocked by previous administration of the ghrelin receptor antagonist ([D-Lys3]-growth hormone-releasing peptide-6). Application of the ghrelin receptor antagonist after ghrelin administration proved ineffective. The results indicate that vasopressin and oxytocin release is influenced directly by the ghrelin system, and the effects of ghrelin on vasopressin and oxytocin secretion from the neurohypophyseal tissue in rats can occur at the level of the posterior pituitary. Our observations lend support to the view that neurohypophysis contains ghrelin receptors.

The effects of hypokalaemia on the hormone exocytosis in adenohypophysis and prolactinoma cell culture model systems.

The extracellular ion milieu determines the exocytosis mechanism that is coupled to spontaneous electrical activity. The K(+) ion plays crucial role in this mechanism: as the potassium current is associated with membrane hyperpolarization and hormone release through protein cascade activation. The primary aim of this study was to investigate the response mechanisms of normal adenohypophysis and adenohypophyseal prolactinoma cell populations at different extracellular K(+) levels with an otherwise isoionic milieu of all other essential ions. We focused on prolactin (PRL) and adrenocorticotrophic hormone (ACTH) release.In our experimental study, female Wistar rats (n=20) were treated with estrone-acetate (150 µg/kg b.w./week) for 6 months to induce prolactinomas in the adenohypophysis. Primary, monolayer cell cultures were prepared by enzymatic and mechanical digestion. PRL and ACTH hormone presence was measured by radioimmunoassay or immuno-chemiluminescence assay. Immunocytochemistry was used to assess the apoptotic cells.Differences between the effects of hypokalaemia on normal adenohypophysis cultures and prolactinoma cell populations were investigated. Significant alteration (p<0.001, n=10) in hormone exocytosis was detected in K(+) treated adenohypophyseal and prolactinoma cell cultures compared to untreated groups. Immunocyto-chemistry showed that Bcl-2 expression was reduced under hypokalaemic conditions.The decrease in hormone exocytosis was tightly correlated to the extracellular K(+) in both cell types, leading to the conclusion that external K(+) may be the major factor for the inhibition of hormone release. The significant increase in hormone content in supernatant media suggests that hypokalaemia may play important role in apoptosis.

Behavioral and endocrine effects of chronic exposure to low doses of chlorobenzenes in Wistar rats.

Chlorobenzenes have often been applied to study persistent organic pollutants with endocrine disruptor effects (POP/EDCs), but with the focus mainly on physiological aspects. Few data exist on the effects of chlorobenzenes and most POP/EDCs on anxiety or other arginine-vasopressin (AVP)- and oxytocin (OXT)-mediated behavior, albeit exposure to POP/EDCs or their ambient mixtures, even in low doses, may pose health risks for subjects living in contaminated areas and/or consuming polluted food. Our primary aim was therefore to demonstrate behavioral effects of longterm exposure to a discrete dose of a chlorobenzene mixture, and to draw attention to the results of subtoxic oral exposure on anxiety-related elements and the possible underlying endocrine processes. Adult male Wistar rats were treated daily with a mixture (ClB) of 1 µg/kg each of hexachlorobenzene and 1,2,4-trichlorobenzene via a gastric tube for 30, 60 or 90 days. After exposure, anxiety-related behavioral elements were determined in open-field and elevated plus maze tests. At euthanasia, the plasma levels of AVP, OXT and adrenocorticotrophic hormone (ACTH) were measured. Simultaneously, pituicytes from subjects were cultured to study the levels of basal and serotonin- or norepinephrinestimulated AVP and OXT secretion. Various anxiety-related behavioral elements were observed to be increased in both tests. The plasma AVP, OXT and ACTH concentrations were increased, to extents depending on the duration of exposure. The basal and monoamine-stimulated levels of AVP and OXT secretion of pituicytes prepared from the ClB-exposed rats were also elevated. Thus, certain anxietyrelated behavioral and endocrine elements were modulated by long-term exposure to ClB. As adult subjects were involved, which are generally less susceptible to toxic agents, it may be concluded that discrete doses of POP/EDC chlorobenzenes that are low enough to fall below the range of legal regulation may exert anxiogenic effects, which suggests that certain anxiogenic disorders may be induced environmentally in exposed human populations.

Further analysis of behavioral and endocrine consequences of chronic exposure of male Wistar rats to subtoxic doses of endocrine disruptor chlorobenzenes.

Many chemicals utilized by humans are present as environmental pollutants and may influence homeostasis from neurological, immunological, endocrinological and/or behavioral aspects. Such agents, acting alone or in ambient mixtures, may be biologically active even at extremely low doses, and it may be postulated that stable, bioaccumulative, reactive endocrine disruptors may affect central and/or peripheral secretion of arginine-vasopressin (AVP) and oxytocin (OXT) and thereby related physiological and behavioral functions, potentially leading to disorders in exposed subjects. The primary aim of this study was to demonstrate effects of chronic exposure to a low dose of an orally administered chlorobenzene mixture on anxiety-related and aggressive behavior mediated largely by AVP and OXT. Chlorobenzenes were applied to model ambient mixtures of endocrine disruptors. Adult, male Wistar rats were exposed daily to 0.1 µg/kg of 1,2,4-trichlorobenzene and hexachlorobenzene via a stomach tube for 30, 60 or 90 days, after which anxiety-related and aggressive behavioral elements were examined in open-field, elevated plus maze and resident-intruder tests. The plasma levels of AVP, OXT and adrenocorticotrophic hormone at the endpoints were measured by radioimmunoassay or immunochemiluminescence assay. The levels of basal and serotonin- or norepinephrine-stimulated AVP and OXT secretion in pituicyte cultures prepared from the posterior lobe of the pituitaries were also measured. The hormone levels proved to be increased to extents depending on the duration of exposure to the chlorobenzenes. Several anxiety-related and aggressive behavioral elements were also enhanced following chlorobenzene exposure, while certain explorative and locomotive elements of the animals were decreased. As both physiological and behavioral elements were modulated by chronic, subtoxic doses of chlorobenzenes, it is concluded that doses of such environmental pollutants low enough to fall outside the range of legal regulation may pose potential risks of anxiogenic and/or aggressive consequences in exposed subjects, including humans.

Effects of galanin-monoaminergic interactions on vasopressin secretion in rat neurohypophyseal cell cultures.

The effects of dopamine (DA), serotonin (5-HT), histamine (HA), adrenaline (ADR), noradrenaline (NADR) and K(+) administration on vasopressin (VP) secretion were studied in 13-14-day cultures of rat neurohypophyseal (NH) cells, and it was examined whether galanin (GAL) can modify the VP release enhancement induced by these monoaminergic compounds. An enzymatic dissociation technique was used to make the rat NH cell cultures. The VP contents of the supernatants of 14-day cultures were determined by radioimmunoassay. Following the administration of 10(-6) M GAL, the VP secretion into the supernatant media decreased. DA, 5-HT, ADR or NADR treatment increased the VP level substantially, while the enhancing effect of HA was more moderate. GAL administration before DA, ADR and NADR treatment prevented the VP concentration increase induced by DA, ADR or NADR. Preincubation with GAL reduced the 5-HT- or HA-induced VP level increases; the VP concentrations of the supernatant media remained above the control level. The GAL blocking effect was prevented by previous treatment with the GAL receptor antagonist galantid (M15). GAL had no effect on the VP level increase induced by K(+), which causes a non-specific hormone secretion. The results indicate that the changes in VP secretion induced by the monoaminergic system can be directly influenced by the GAL-ergic system. The interactions between the monoaminergic and GAL-ergic systems regarding VP secretion occur at the level of the posterior pituitary.

Significance of the adrenergic system in the regulation of vasopressin secretion in rat neurohypophyseal tissue cultures.

UNLABELLED: The effects of adrenaline (A) and noradrenaline (NA) on vasopressin (VP) secretion were studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP contents of the supernatant media were determined by radioimmunoassay after a 1 or 2-h incubation. Significantly increased VP levels were detected in the tissue culture media following the administration of A (an alpha+beta(2)-receptor agonist), depending on the dose of A. The VP secretion elevation was totally blocked by the previous administration of phentolamine (an alpha(1)+alpha(2)-receptor antagonist) or corynanthine (an alpha(1)-receptor antagonist). Yohimbine (an alpha(2)-receptor antagonist) did not influence the VP secretion increase induced by A. After the administration of NA (a beta+alpha(1)-receptor agonist), a VP secretion elevation was again detected, but the degree of enhancement proved smaller than that of the VP secretion increase induced by A. Propranolol (a beta(1)+beta(2)-receptor antagonist) before NA administration prevented the VP secretion increase. Atenolol (a beta(1)-receptor antagonist) did not block the VP secretion elevation induced by NA. Corynanthine (an alpha(1)-receptor antagonist) treatment before NA administration reduced the NA-induced VP enhancement, because NA has an alpha(1)-receptor agonist character in addition to its main character (a beta-receptor agonist). Surprisingly, the administration of pindolol (a beta(1)+beta(2)-receptor antagonist) enhanced VP secretion. This contradictory effect can be explained in that pindolol not only acts as a blocker, but also exerts "intrinsic sympathomimetic action" and a strong adrenergic agonist effect. Pindolol before NA administration significantly increased the NA-induced VP elevation. CONCLUSIONS: Mainly the alpha(1)- and beta(2)-adrenergic receptors are involved in the A- or NA-induced increase of VP secretion in isolated NH tissue cultures. The results indicate that VP release is influenced directly by the adrenergic system, and the adrenergic control of VP secretion from the NH tissue in rats can occur at the level of the posterior pituitary.

Histamine-induced enhancement of vasopressin and oxytocin secretion in rat neurohypophyseal tissue cultures.

The effects of histamine (HA) on vasopressin (VP) and oxytocin (OT) secretion were studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP and OT contents of the supernatant were determined by radioimmunoassay (RIA) after a 1 or 2-h incubation. Significantly increased levels of VP and OT production were detected in the tissue culture media following HA administration, depending on the HA dose. The elevation of NH hormone secretion could be partially blocked by previous administration of the HA antagonist mepyramine (MEP, an H1 receptor antagonist) or cimetidine (CIM, an H2 receptor antagonist). Thioperamide (TPE, an H3-H4 receptor antagonist) did not influence the VP or OT secretion increase induced by HA. The application of MEP, CIM or TPE after HA administration proved ineffective. The H1 and H2 receptors are mainly involved in the HA-induced increase of both VP and OT secretion in isolated NH tissue cultures. The results indicate that NH hormone release is influenced directly by the histaminergic system, and the histaminergic control of VP and OT secretion from the NH tissue in rats can occur at the level of the posterior pituitary.

Serotonin-induced enhancement of vasopressin and oxytocin secretion in rat neurohypophyseal tissue culture.

The effects of serotonin (5-hydroxytryptamine; 5-HT) on vasopressin (VP) and oxytocin (OT) secretion were studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP and OT contents of the supernatant were determined by radioimmunoassay after a 1 or 2 h incubation. Significantly increased levels of VP and OT production were detected in the tissue culture media following 5-HT administration, depending on the 5-HT dose. The elevation of NH hormone secretion could be partially blocked by previous administration of the 5-HT antagonist ketanserin or metergoline. WAY-100635 did not influence the increased VP secretion induced by 5-HT, but the elevated OT production was prevented by WAY-100635 before 5-HT administration. The application of WAY-100635, ketanserin or metergoline, after 5-HT administration proved ineffective. The results indicate that NH hormone release is influenced directly by the serotonergic system. The serotonergic control of VP and OT secretion from the NH tissue in rats can occur at the level of the posterior pituitary.

Inhibitory effect of galanin on dopamine induced increased oxytocin secretion in rat neurohypophyseal tissue cultures.

The regulation of oxytocin (OT) release by galanin (GAL) at the neurohypophyseal (NH) nerve terminal is not adequately understood. The effect of GAL on the secretion of OT was studied in 13- to 14-day cultures of isolated rat NH tissue. By this time, the hormone content of the medium had become constant. The OT content of the supernatant medium was determined by RIA after a 1- or 2-h incubation. A significantly decreased content of OT was found following incubation with 10(-6)-10(-8) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the OT synthesis of NH tissue cultures. This elevation of OT secretion could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that OT release from the NH is directly influenced by the GAL-ergic system. The GAL-ergic control of OT secretion from NH tissue in rats can occur at the level of the posterior pituitary.

Inhibitory effect of galanin on dopamine-induced enhanced vasopressin secretion in rat neurohypophyseal tissue cultures.

The effect of galanin (GAL) on vasopressin (VP) secretion was studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP content of the supernatant was determined by radioimmunoassay (RIA) after a 1- or 2-h incubation. A significantly decreased content of VP was detected following the administration of 10(-6)-10(-9) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the VP level of NH tissue cultures. This VP concentration elevation could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that VP release is directly influenced by the GAL-ergic system. The GAL-ergic control of VP secretion from NH tissue in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Gastric mucosal endogenous prostacyclin levels are different in Brattleboro rats compared with Wistar strain.

Homozygous Brattleboro rats were investigated and compared to normal (physiological) Wistar strain rats regarding their gastric mucosal endogenous prostacyclin (PG-I(2)) level. It seems that the Brattleboro animals have a significantly lower level of this important protective material. Wistar rats having an artificial pituitary stalk lesion (which is the artificial equivalent of homozygous Brattleboro animals) showed no differences in endogenous mucosal prostacyclin level compared to normal Wistar rats. Therefore, we concluded that this hitherto unknown property of the homozygous Brattleboro rats is genetically determined.

On a possible new intracellular signal-system in rat gastric mucosa.

It is known that cAMP and cGMP, as an "intracellular second messenger system" play a significant role as a signal system, in the mechanism of action of anti-ulcerogenic (cytoprotective) drugs. According to our present, preliminary investigations it seems that during different experimental circumstances the gastric mucosal 3'-5'-cyclic-cytidine-mono-phosphate (cCMP) 3'-5'-cyclic-uridine-monophosphate and (cUMP) levels were changed--similarly to CAMP and cGMP--and these changes might be a possible indicator of a further, most probably secondary, signal- system role.

Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures.

The effects of dopamine (DA) or DA-active drugs on the synthesis of neurohypophyseal (NH) hormones were studied in 13-14 day cultures of isolated NH tissue from rats. The following DA-active compounds were used (10(-6) M in each medium): DA, apomorphine (APM), Pro-Lys-Gly (PLG), butaclamol (B), haloperidol (HP), chlorpromazine (CPZ) and sulpiride (SP). The oxytocin (OT) and vasopressin (VP) contents of the condensed media were determined by RIA after a 1 or 2 h incubation. Significantly increased contents of OT and VP were detected in the tissue culture media following DA, APM or PLG administration. This elevation of NH hormone production could be blocked by previous administration of B or the DA receptor antagonists HP, CPZ or SP. The application of B after DA agonists proved ineffective. The results indicate that NH hormone production can be directly influenced by the DA-ergic system. The DA-ergic control of NH hormone secretion in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats.

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.

The effect of osmolality changes on gastric mucosal endogenous prostacyclin levels in drug-induced experimental ulcer model of rat.

Osmolality changes evoked with intragastric administration of natural honey or mannitol, significantly decreased the gastric ulceration of rats induced by indomethacin. Together with this effect, a parallel increase was detectable in the mucosal level of endogenous prostacyclin. Although many processes may be involved in this phenomenon, the authors explain their data with a stimulating effect on gastric mucosal microcirculation due to osmolality changes.

Primary mono-layer cell cultures as model system for studying of environmental toxic agents: organochlorine compounds.

Organic pollution of water and soil has various harmful effects on biological systems (1). Chlorine substituted benzol compounds are one these xenobiotic substances, which are toxic to the environment (2). They can also accumulate in plant and animal tissues (3), which provides ample reason to study the effects of sublethal doses of chloro-benzols on various cell cultures. In this study the toxic effects of chloro-benzols were investigated on avian fibroblast and mammalian hepatocyte cultures. The fibroblast cultures were prepared from eggs preadapted to chloro-benzol during a fourteen-day-long incubation period. The Wistar rat hepatocyte monolayer cultures were exposed to a direct treatment of 1,2,4-tri-chloro-benzol (0.01 microgram/ml-1 microgram/ml) for 3 hours. Following the treatment with chloro-benzol, the viability of the cells was measured, together with lactic dehydrogenase activity, in both kinds of cultures. The effect of tri-chloro-benzol treatment on chicken eggs was not significant. The cells of chicken embryos were not damaged after the 1,2,4-tri-chloro-benzol treatment. The hepatocyte cultures showed the toxic effects of 1,2,4-tri-chloro-benzol after the direct and acute treatment. The cell viability decreased and the LDH activity increased significantly. These results show that the primary cell cultures are suitable for studying the effects of organochlorine compounds.

Functional membrane changes due to tumor induction in rat pituitary cell cultures.

Membrane functions in tumorous cells are different from those in healthy cells. The aim of the present study was to investigate the changes in pituitary cell membrane functions and hormone secretion after tumor induction in vivo and in vitro. Prolactinomas were induced in vivo in female Wistar rats with estrone acetate. Normal anterior pituitaries and prolactinomas of female Wistar rats were dissociated enzymatically and mechanically, then cultured on collagen-treated plastic dishes. Some normal anterior pituitary cultures were treated with benz(c)acridines as tumorigenic agents in vitro. Intracellular 3',5'-cyclic-adenosine monophosphate (cAMP) levels were determined by a competitive binding technique, membrane fluidity was assayed by fluorescence anisotropy, and ATP-ase activities were estimated via ATP loss. The results indicated decreased membrane fluidity in tumorous cell cultures. However, in vitro benz(c)acridine treatment exerted more pronounced effects than those observed after in vivo estrone treatment. The ATP-ase activities were highly increased in benz(c)acridine-treated cells and in estrogen-induced prolactinoma cells, more strongly so in the former ones. The intracellular cAMP levels were higher than normal in both of them. The results concerning the ACTH, alpha-MSH, PRL and GH levels of normal and tumorous cell cultures were published in our previous study. Our findings show that the tumorous transformation of pituitary cells can cause significant changes in functional membrane parameters and hormone secretion. Decreased membrane fluidity was accompanied by an increased exocytosis (hormone release) and adenylate cyclase activity in tumorous cells.

Surface changes and hormone production in pituitary cells during tumorigenesis.

Prolactinomas were induced by chronic estrone acetate treatment in female Wistar rats in vivo. After enzymatic dissociation of the tumor tissue, monolayer cell cultures were obtained. In vitro tumor induction was performed by treatment of normal monolayer anterior pituitary cell cultures with a 1:1 mixture of 7,9-dimethylbenz[c]acridine: 8-methylbenz[c]acridine. For immunohistochemistry, the cell cultures were stained by the peroxidase-antiperoxidase method for standardization; the nonspecific hormone release was induced with 30 mM K+. The prolactin, alpha-melanotropin, and adrenocorticotropin levels in the supernatant were measured by specific, sensitive radioimmunoassays. The results indicated surface differences between the in vivo induced prolactinoma cells and normal pituitary cells: the attachment of the prolactinoma cells required 15% collagen treatment, whereas normal cells required only 3-4% collagen. Tumor cells induced in vitro by methylbenz[c]acridine treatment were able to attach only after ammonia activation of the collagen surface. These findings strongly suggest that the mode of tumor induction can result in differences in membrane fluidity; this phenomenon is possibly connected with the levels of prolactin, adrenocorticotropin and alpha-melanotropin hormone production of these endocrine tumor cells.

Identification of oxytocin and vasopressin from neurohypophyseal cell culture.

Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary. The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay. By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

Adenomatous leydig cell hyperplasia of the testicle remnant after subtotal orchidectomy in the rat.

Subtotal orchidectomy is a suitable method to induce adenomatous Leydig cell hyperplasia in the testicular remnant without irradiation or toxic chemicals. Very expressed hyperplasia of the smooth endoplasmic reticulum was detectable electronmicroscopically in the newly formed Leydig cells. The activity of Δ5-3ß-hydroxysteroid dehydrogenase in the homogenate of the testicular remnant was found considerably elevated. These observations suggest a steroidogenetic activity of the proliferating Leydig cells.