Analysis of the effect of neuropeptides and cannabinoids in gastric mucosal defense initiated centrally in the rat.

Increasing number of evidence suggest that gastric mucosal protection can be induced also centrally. Several neuropeptides, such as TRH, amylin, adrenomedullin, enkephalin, beta-endorphin, nociceptin, nocistatin, ghrelin or orexin given centrally induce gastroprotection and the dorsal vagal complex and vagal nerve may play prominent role in this centrally initiated effect. Since also cannabinoid receptors are present in the dorsal vagal complex, we aimed to study whether activation of central cannabinoid receptors result in gastric mucosal defense and whether there is an interaction between cannabinoids and endogenous opioids. Gastric mucosal damage was induced by 100% ethanol in rats. The cannabinoids were given intravenously (i.v.) or intracerebroventricularly (i.c.v.), while the antagonists were given i.c.v or intracisternally (i.c.). Gastric lesions were evaluated macroscopically 60 min later. Anandamide, methanandamide and WIN55,212-2 reduced ethanol-induced mucosal lesions after both peripheral (0.28-5.6 micromol/kg, 0.7-5.6 micromol/kg and 0.05-0.2 mumol/kg i.v., respectively) and central (2.9-115 nmol/rat, 0.27-70 nmol/rat and 1.9-38 nmol/rat i.c.v., respectively) administration. The gastroprotective effect of anandamide and methanandamide given i.c.v. or i.v.was reversed by the CB(1) receptor antagonist SR141716A (2.16 nmol i.c.v.). Naloxone (27.5 nmol i.c.v.) also antagonized the effect of i.c.v. or i.v. injected anandamide and WIN55,212-2, but less affected that of methanandamide. The gastroprotective effect of anandamide was diminished also by endomorphin-2 antiserum. In conclusion it was first demonstrated that activation of central CB(1) receptors results in gastroprotective effect. The effect is mediated at least partly by endogenous opioids.

Maternal genotype influences stress reactivity of vasopressin-deficient brattleboro rats.

The role of vasopressin, cosecreted with corticotropin-releasing hormone (CRH), in stress is debated, because both normal as well as reduced adrenocorticotropin hormone (ACTH) rise to an acute challenge has been reported in Brattleboro rats genetically lacking vasopressin (di/di). Because di/di pups could be born either from di/+ (heterozygous) or from di/di mothers, and maternal influence is known to modify adult responsiveness, we investigated whether the influence of maternal genotype could explain the variability. Adult rats from mothers with different genotypes were stressed with 60 min restraint and trunk blood was collected for measuring hormone content by radioimmunoassay at the end of stress. All offspring of di/+ mothers had similar ACTH responses to restraint, while the di/di rats born to, and raised by di/di mothers showed reduced ACTH reactivity to restraint. The di/di rats showed elevated water turnover and required a daily cage cleaning every day, which meant frequent handling. To offset the role of handling, all rats had daily cage cleaning in the next series, but the results were the same as in the first series. To investigate whether lactation, the behaviour of the mother or some other factor during the pregnancy is responsible for the differences, pups from di/+ dams were raised by di/di foster mothers and vice versa. We found that the genotype of parental mother is more important than that of the foster mother. The corticosterone and prolactin elevation normally seen after acute stress was unchanged by family history, maternal or personal genotype. Furthermore, in studies with mutant animals, the rearing conditions should be controlled by the experimenter. In experiments with Brattleboro rats, the use of homozygous and heterozygous rats from the same litters of di/+ dams and di/di males is recommended. Our results suggest that vasopressin is not indispensable for ACTH release, and that the di/di genotype of the parental mother can decrease the stress reactivity of the di/di Brattleboro rats.

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint.

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.

Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis.

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.

Growth hormone secretion and activation of cyclic AMP by growth hormone releasing hormone and gamma-aminobutyric acid in the neonatal rat pituitary.

The effect of factors influencing pituitary growth hormone secretion may be mediated by a combination of several intracellular mechanisms. The involvement of cyclic AMP (cAMP) in the GH stimulatory effect of tau-aminobutyric acid (GABA) and of growth hormone releasing hormone (GHRH) was studied in neonatal rat pituitaries. In the pituitaries of the newborn rats GH secretion was stimulated by forskolin and by isobutylmethylxantine (IBMX). GHRH but not GABA elevated pituitary cAMP concentration, whereas both drugs increased GH secretion from 2-day old pituitaries. IBMX did not augment the cAMP stimulating effect of GHRH in 2-day old, but potentiated it in older (7, 14 and 21-day old) pituitaries. The results indicate the presence of a functioning, but relatively immature intracellular signal transmission system in the 2-day old rat pituitary.

gamma-Aminobutyric acid-induced elevation of intracellular calcium concentration in pituitary cells of neonatal rats.

Gamma-aminobutyric acid (GABA) increased intracellular calcium concentration ([Ca2+]i) of newborn rat pituitary cells in suspension measured by the FURA-2 method. The effect of GABA was dose dependent in the range of 0.1-10 microM. This effect diminished with postnatal age as measured at days 2, 14 and 21, and in adult animals. The GABA stimulation was mimicked by muscimol; in contrast, baclofen (up to 100 microM) was ineffective. Picrotoxin, a GABAA antagonist interacting with GABA-activated chloride ionophores, caused a dose-dependent inhibition of the [Ca2+]i elevating effect of 100 microM GABA or muscimol. These observations indicate the involvement of GABAA type receptors. The GABA or muscimol effect on [Ca2+]i was antagonized by nifedipine (10 microM) or verapamil (50 microM), and completely abolished in the presence of 4 mM EGTA (low-calcium medium). The findings indicate the presence of depolarizing GABAA receptors on neonatal rat pituitary cells. It seems very likely that the mechanism by which GABA receptor occupation results in elevated [Ca2+]i is a membrane depolarization by increased Cl- conductance followed by calcium influx through L-type voltage-dependent calcium channels.

Significance of chloride channel activation in the gamma-aminobutyric acid induced growth hormone secretion in the neonatal rat pituitary.

Growth hormone (GH) secretion of the neonatal pituitary is stimulated by tau-aminobutyric acid (GABA) (1,2). Since in most cases GABA is known to act by increasing postsynaptic membrane permeability to chloride ions we tested the importance of chloride channel activation in the GH stimulatory effect of GABA in the neonatal pituitary. In the absence of chloride in the superfusion medium GABA was without effect on GH secretion of the neonatal pituitaries and its effect was attenuated by chloride channel inhibitors. The effect of growth hormone releasing hormone (GHRH) on GH secretion was attenuated in the chloride-free media, but it was not affected by simultaneous administration of chloride channel blockers. The present study indicates that GH stimulatory effect of GABA in the neonatal pituitaries might involve chloride channel activation probably resulting in secondary activation of calcium channels.

Evidence for the localization of hydrogen peroxide-stimulated cyclooxygenase activity in rat brain mitochondria: a possible coupling with monoamine oxidase.

The distribution of basal and of H2O2-stimulated cyclooxygenase activity in the primary fractions of rat brain homogenates and in the subfractions of crude mitochondrial fraction was studied. For comparison, the localization of H2O2-generating monoamine oxidase (MAO) as well as that of the mitochondrial marker succinate dehydrogenase (SDH) was also examined. H2O2 was generated by MAO using 5 x 10(-4) M noradrenaline (NA) or 2 x 10(-4) M 2-phenylethylamine (PEA) as substrates, or by 25 micrograms glucose oxidase (GOD) per ml in the presence of 1 mM glucose. For nonstimulated (basal) cyclooxygenase, the relative specific activity (RSA) was high in microsomes (1.79) and in the free mitochondria-containing subfraction of the crude mitochondrial fraction (1.94). Parallel distribution of MAO and H2O2-stimulated cyclooxygenase was observed in all fractions studied in the presence of NA. The highest RSA was found in the purified mitochondria for both enzymes (1.85 for MAO and 1.97 for H2O2-stimulated cyclooxygenase). The enrichment of SDH (RSA = 2.21) indicated a high concentration of mitochondria in this fraction. The same distribution of H2O2-stimulated cyclooxygenase was obtained when, instead of the MAO-NA system, hydrogen peroxide was generated by GOD in the presence of glucose. H2O2 generated by deamination of NA or PEA by MAO, or during the enzymatic oxidation of glucose by GOD, caused a threefold increase in mitochondrial endoperoxide formation. Indomethacin (2 x 10(-4) M), catalase (50 micrograms/ml), and pargyline (2 x 10(-4) M) eliminated the MAO-dependent mitochondrial synthesis of PG endoperoxides. The GOD-dependent cyclooxygenase activity in this fraction was abolished by indomethacin or catalase, but not by pargyline. The results show the existence of a mitochondrial cyclooxygenase in brain tissue. The enzyme is sensitive to H2O2 and produces prostaglandin endoperoxides from an endogenous source of arachidonic acid. The identical localization of H2O2-producing MAO and H2O2-sensitive cyclooxygenase suggests a possible coupling between monoamine and arachidonic acid metabolism.

On the mechanism of the involvement of monoamine oxidase in catecholamine-stimulated prostaglandin biosynthesis in particulate fraction of rat brain homogenates: role of hydrogen peroxide.

The mechanism of involvement of monoamine oxidase (MAO) in catecholamine-stimulated prostaglandin (PG) biosynthesis was studied in the particulate fraction of rat brain homogenates. High concentrations of either noradrenaline (NA) or dopamine (DA) stimulated effectively PGF2 alpha formation. The same amount of 2-phenylethylamine (PEA) acted similarly, provided that it was administered together with a catecholamine analogue or metabolite possessing the 3,4-dihydroxyphenyl nucleus--3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylglycol (DOPEG), 3,4-dihydroxyphenylacetaldehyde (DOPAL), or alpha-methylnoradrenaline (alpha-met-NA)--or with SnCl2. In the absence of PEA, these compounds were ineffective with regard to stimulation of PGF2 alpha formation. Catalase, pargyline, or indomethacin abolished completely PGF2 alpha formation elicited either by catecholamines or by PEA plus a 3,4-dihydroxyphenyl compound or SnCl2. With regard to the stimulation of PGF2 alpha formation in the presence of alpha-met-NA, PEA could be replaced by H2O2 generated by the glucose oxidase(GOD)-glucose system. The effect of H2O2 was inhibited by indomethacin or catalase, but pargyline was ineffective. It is assumed that catecholamines play a dual role in the activation of PG biosynthesis in brain tissue. During the enzymatic decomposition of catecholamines MAO produces H2O2, which stimulates endoperoxide synthesis. Simultaneously, catecholamines as hydrogen donors promote the nonenzymatic transformation of endoperoxides into PGF2 alpha. The possible physiological importance of these findings is discussed.