Molecular differentiation of four Reptalus species (Hemiptera: Cixiidae).

The cixiid species Reptalus quinquecostatus, R. cuspidatus, R. panzeri and R. melanochaetus are widely distributed in Europe and are receiving growing attention because of their potential role as phytoplasma vectors. Identifying the Reptalus species is restricted to a few specialist entomologists and relies on the morphology of the male genitalia, hampering the identification of juveniles and females. This study provides the tools for species discrimination by integrating the morphological description, which is primarily for the genus identification, with new molecular assays, based on both ribosomal and mitochondrial DNA. PCR-RFLP assays carried out on the mitochondrial cytochrome oxidase I gene (COI) with AluI provided species-specific profiles for the four Reptalus species. Amplification of a ribosomal internal transcribed spacer (ITS2) region produced species-specific fragments of different sizes for R. quinquecostatus, R. melanochaetus, R. cuspidatus and R. panzeri. The digestion of the ITS2 PCR product with TaqI allowed the discrimination of these latter two species. This molecular identification key ensures reliable results and can be successfully applied not only to adults, but also to the nymphs feeding on the roots. The identification of the nymphs (i) extends the collection period of these monovoltine species to the whole year (adults are present for a short summer period) and (ii) allows the unambiguous identification of their actual host plants because nymphs are steady on the root system while adults tend to disperse onto other plants. Fast and reliable identification of the Reptalus species provides useful help in monitoring activities and, therefore, in designing rational control strategies to protect crops from phytoplasma infection.

Comparison on DNA patterns of different ecotypes of European corn borer (Ostrinia nubilalis Hübner) in Hungary.

For a molecular genetic study on Hungarian populations of European corn borer L5 stage larvae were collected from 14 places of three different regions of the country (uni- and bivoltine ecotypes). Additionally, the study included larvae from Egypt, too (multivoltine ecotype). Molecular examinations of European corn borer larvae using the study of mitochondrial cytochrome b (cyt b) revealed that by single strand conformation polimorphism (SSCP) the populations found in Hungary represented the same haplotype. Even the Egyptian sample showed no genetic divergence. Some minor deviatons were found in the case of a sample from Székkutas, but that did not prove the genetic divergence of the bivoltine ecotype either, since the other samples of South-East Hungary did not display this kind of genetic variation. On the basis of our investigations it can be said that the univoltine and bivoltine generations, have uniform genetic complements.

The theoretical basis for scaling-up by the use of the method of microwave granulation.

For the scaling-up achieved by the use of the method of wet microwave granulation based on calculations, there is a need for an exact mathematical description for the relationship between the dose of radiation and the resultant effect. By assessing the physical, physical-chemical, and chemical factors, we may conclude that, by the gross kinetic evaluation of the change in the enthalpy and the loss of humidity, there is a possible solution for the mathematical description of the single-step, single-pot granulation from the practical aspect of finding ways to scale-up. This paper overviews the experiments performed in a laboratory-size microwave vacuum granulator in testing two different granules with respect to composition and permittivity and presents the evaluation of the experimental data.

Effect of cholinergic drugs on the concentration of intracellular free calcium of rat pituitary intermediate lobe cells.

We tested the effect of cholinergic drugs on the concentration of intracellular free calcium in rat melanotropes. Acetylcholine, muscarine, carbachol, and nicotine resulted in a significant rise in this parameter. Effect of acetylcholine was reduced by atropine (non-selective muscarinic antagonist), pirenzepine (M1 muscarinic antagonist), and 4-DAMP (M3 > M1 muscarinic antagonist), but exposure to the M1 muscarinic agonist McN-A 343 resulted in a significantly smaller calcium-response than that seen in response to acetylcholine or to muscarine. This suggests the involvement of both M1 and M3 muscarinic receptors in the acetylcholine-induced calcium-rise. On the other hand, in the presence of atropine the acetylcholine-induced calcium-rise was not eliminated: this fact indicates that nicotinic receptors are also involved in the acetylcholine-induced intracellular calcium-rise. The acetylcholine-, and nicotine-induced calcium-rise was significantly reduced in presence of the neuronal-type nicotinic antagonist, mecamylamine. This suggests the involvement of a neuronal-type nicotinic receptor in the acetylcholine-induced intracellular calcium-response. Moreover, because in a further experiment almost 80% of the cells investigated responded to muscarine as well as nicotine, we conclude that both functionally active muscarinic and nicotinic receptors are present on the same cell.

Monosodium glutamate lesions inhibit the N-methyl-D-aspartate-induced growth hormone but not prolactin release in rats.

Large doses of glutamate administered to newborn rats damage permanently the neurones in the hypothalamic arcuate nucleus containing the growth hormone releasing hormone and the prolactin inhibiting dopamine neuron cell bodies. Since adult animals that underwent neonatal glutamate treatment still have a relatively well functioning growth hormone and prolactin system, we tested whether in the adults the excitatory amino acid sensibility is changed. After i.v. injection of different doses (10 or 30 mg/kg) of N-methyl-D-aspartate (excitatory amino acid receptor subtype agonist) growth hormone levels were significantly increased in the control groups but there was no rise in neonatally glutamate treated male and female rats. The level of prolactin was increased by N-methyl-D-aspartate, too, but the glutamate treatment had no effect on the rise. Our study suggests that systemic administration of N-methyl-D-aspartate increases plasma growth hormone level by activating the growth hormone releasing cells in the arcuate nucleus, but the intact tuberoinfundibular dopaminergic pathway is not essential for its prolactin stimulatory effect.

Catecholaminergic control of intracellular free calcium and beta-endorphin secretion of rat pituitary intermediate lobe cells.

Individual melanotropes and intermediate lobes were tested to elucidate the role of alpha- and beta-adrenergic and D-2 dopamine receptors in the regulation of concentration of intracellular free calcium ([Ca2+]i) and release of beta-endorphin. Hormone secretion was studied in a superfusion system, while [Ca2+]i was measured microspectrofluorimetrically. Noradrenaline (1 microM) resulted in a slight decrease, then a marked increase in [Ca2+]i and secretion of beta-endorphin. The nonselective beta-adrenergic agonist isoproterenol (1 microM) increased [Ca2+]i and secretion of beta-endorphin; this effect was blocked by the beta-antagonist propranolol (10 microM). The alpha-adrenergic agonist phenylephrine (1 microM) increased [Ca2+]i and beta-endorphin secretion, but this effect was not blocked by terazosin or prazosin (alpha1-adrenergic antagonists, 1 microM). Administration of the alpha2-adrenergic agonist xylazine (1 microM) increased [Ca2+]i but did not affect secretion of the hormone. Biphasic effect of noradrenaline was tested in presence of adrenergic and dopaminergic antagonists. The noradrenaline-induced rise in [Ca2+]i and beta-endorphin secretion was decreased by propranolol, but this drug did not modify the inhibition. In the presence of 1 microM sulpiride (selective D-2 dopaminergic antagonist), the inhibitory phase of the curve was abolished, and the subsequent increase was reduced. This suggests that activation of dopamine D-2 receptors is involved not only in the inhibition, but also in the subsequent increase, which may originate from a rebound after the termination of the activation of these inhibitory receptors. Our data suggest the presence of several distinct types of catecholamine receptors in the rat intermediate lobe, and the dominant involvement of D-2 and beta-adrenergic receptors in the noradrenaline-induced regulation of melanotropes.

Age-dependent muscarinic stimulation of beta-endorphin secretion from rat neurointermediate lobe in vitro.

The effect of acetylcholine on the neurointermediate lobe beta-endorphin secretion was studied in the neonatal and in the adult rat in vitro. Acetylcholine stimulated beta-endorphin secretion from the 2-day- and 5-day-old neurointermediate lobe, the effect was dose dependent and more pronounced in the presence of the cholinesterase inhibitor eserine. The 10-day-, the 21-day-old and the adult rat neurointermediate lobes did not respond to acetylcholine, even in the presence of eserine. Basal beta-endorphin secretion was elevated by the D2 receptor antagonist sulpiride, but acetylcholine was without effect in the 10-day-old and in the adult neurointermediate lobe even after dopamine receptor blockade. The beta-endorphin stimulatory response to acetylcholine was diminished by the M1 muscarinic receptor antagonist pirenzepine and blocked by the M3 > M1 antagonist 4-diamino-phenyl-piperidine (4-DAMP). The selective M2 antagonist methoctramine and nicotine had no effect. These data indicate that the neurointermediate lobe beta-endorphin secretion is under special muscarinic cholinergic regulation for a relatively short time after birth. The disappearance of this stimulatory cholinergic effect in later life might be due to changes in the intracellular secretory machinery in the IL and/or to the uncoupling of the cholinergic receptors from the intracellular signal transduction system(s) responsible for the stimulated secretion in the rat melanotrope cells.

Acetylcholine stimulates growth hormone secretion in the neonatal rat pituitary.

The direct effect of acetylcholine on pituitary growth hormone secretion during the postnatal period of the rat was studied using a superfusion system. Acetylcholine elicited a dose related stimulatory effect on growth hormone (GH) secretion in the pituitaries from 2-day old rats. M1 muscarinic agonist McN A343 mimicked the GH releasing effect of acetylcholine, nicotine was ineffective. The GH release elicited by acetylcholine diminished with postnatal development, it was small by the end of the third postnatal week and was not demonstrable in the adult pituitaries. The effect of acetylcholine was potently antagonized by pirenzepine (M1 antagonist) and 4-DAMP (M3 and M1 antagonist) but not by methoctramine (M2 antagonist). It is concluded that unlike in the adult, in the newborn rat the cholinergic regulation of pituitary GH secretion plays a prominent role directly at pituitary level most likely via M1 receptors.

Effect of interleukin-1 beta on plasma ACTH, beta-endorphin, and corticosterone levels in infant and prepubertal rats.

IL-1 beta is known to enhance ACTH release from the anterior pituitary in the adult rat, mainly by simulating the hypothalamic ACTH-releasing hormone (CRH) release, but it seems to have a direct effect on the pituitary and on the adrenal hormone secretion, too. The effect of IL-1 beta on the beta-endorphin (beta E) secretion from the intermediate lobe is less well studied. There is very little information on the effect of IL-1 beta on the hypothalamic-pituitary-adrenal axis (HPAA) in the postnatal rat, which is a special period, because the reactivity of the HPAA is blunted. The effect of IL-1 beta in this period seemed to be of special interest, because neither the immune nor the endocrine system is fully developed. In the present study we tested the 30- and 120-min effect of intraperitoneally administered 0.5 and 100 ng/g body weight IL-1 beta on the plasma immunoreactive (ir) ACTH, beta E, and corticosterone (CS) levels in the 10-d-old (infant) and 30-d-old (prepubertal) rat. Generally, the ir-ACTH, ir-beta E, and ir-CS levels were significantly higher in prepubertal than in infant rats. Hormone levels were more enhanced by the higher dose of IL-1 beta, and changes were more pronounced at 120 min than at 30 min. The relative increase of ir-ACTH and ir-beta E was smaller in the infant than in the prepubertal rat. In contrast, the relative increase of ir-CS was more pronounced in the infant rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin-synthesizing and prolactin-releasing activity of fetal and early postnatal rat pituitaries: in vivo and in vitro studies using RIA, reverse hemolytic plaque assay and immunocytochemistry.

In vivo and in vitro prolactin (PRL)-synthesizing and PRL-releasing activity of fetal (days 12-22) and early postnatal (days 1-10 after birth) rat pituitaries were studied by means of radioimmunoassay (RIA), reverse hemolytic plaque assay and immunocytochemistry. Using RIA, PRL could first be detected, both in the pituitary and in the serum, on day 17 of fetal development. From this day on, pituitary PRL gradually increased, the rise was particularly marked during the postnatal period and became depressed for the first 10 days of postnatal life. On fetal day 18, 12-15% of monodispersed pituitary cells displayed PRL immunopositivity, but only 3-5% of PRL-positive cells were plaque-forming, i.e. released PRL. By the end of gestation 19-25% and on postnatal day 10 42-45% of all pituitary cells were PRL cells and 31-35 and 15-17% of PRL-positive cells, respectively released PRL. Both pre- and postnatal PRL cells in monolayers were insensitive to TRH treatment. Pituitary primordia immunocytochemically and radioimmunologically negative for PRL (13- to 14-day-old fetal) when placed in serum-free organ culture were able to synthesize and release PRL. Fetal pituitary exhibited a highly regular increasing pattern of daily PRL release during a 7-day-culture period. Data obtained both in vivo and in vitro did not exhibit any sex differences. The present findings are consistent with all those observations suggesting an early emergence of fetal rat pituitary lactotrophs. The in vitro results support the concept that Rathke's pouch cells have substantial degree of independence from extrapituitary regulatory actions in the expression and further progression of specific functions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Corticotroph, somatotroph and mammotroph cell kinetics in the postnatal infant female rat.

The aim of this work was to detect if hypothalamic-pituitary maturation was accompanied by significant proliferation changes in differentiated pituitary cell pools. For this purpose, pituitary corticotroph (Ct), mammotroph (Mt) and somatotroph (St) proliferation activities were scanned in intact female rats during the postnatal (P) period (1-35 postnatal days). The techniques of tritiated thymidine labelling, immunostaining and autoradiography were combined to visualize DNA synthesis of hormone containing cells. Immunoreactive cell densities were measured using image analysis, and double labelled cells were counted. Corticotroph proliferation activity increased significantly on day P12, followed by an increase in the Ct proportion on days P13-14. This is the first observation of a spontaneous change of corticotroph proliferation at the end of the stress nonresponsive period. The mammotroph density and proliferation rate increased gradually during postnatal maturation, until the Mt pool overran other cell types of the female hypophysis on day 35. The somatotroph pool was the most numerous until day P20; the proliferation rate remained constant while St proportions increased reaching a plateau between days P13 and 20, then decreased to the adult level. Each cell type examined showed a characteristic, individual density and proliferation pattern.

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.

Effects of Hypnorm (fentanyl) on ACTH/beta-endorphin levels in plasma, pituitary and brain of 10-day old rats.

Administration of Hypnorm, an anaesthetic containing the known mu-opiate receptor agonist fentanyl, elicited dose- and time-related elevation of plasma ACTH, beta-endorphin and corticosterone levels in 10-day old rat pups. Pretreatment with specific antibodies (raised against CRH, AVP and ACTH resp.) revealed that Hypnorm administration activated the ACTH-corticosterone system in the 10-day old rat and its effect is mediated by CRH and/or AVP. Hypnorm anaesthesia was associated with significant decrease in the ACTH and beta-endorphin levels in the pituitary lobes as well as in beta-endorphin content of the hypothalamus and medulla oblongata. Latter results may indicate that the beta-endorphinergic system in the brain of the 10-day old rat is activated by Hypnorm, an effect most probably elicited by the opiate agonist fentanyl.

Possible mediation of GABA induced growth hormone secretion by increased calcium-flux in neonatal pituitaries.

Gamma-aminobutyric acid (GABA) stimulates growth hormone (GH) secretion from pituitaries of young (less than 20-day old) rats (1,2). Present work revealed that the GH stimulatory effect of GABA was abolished in the absence of calcium and response was attenuated by Nifedipine. The calcium efflux from 45CaCl2 preloaded neonatal pituitaries was enhanced by GABA or by muscimol, and this effect was antagonized by the GABA antagonist picrotoxin. In pituitaries of 21 day old or adult rats GABA stimulated neither GH secretion nor calcium efflux. These results indicate that in neonatal pituitaries GABA influences calcium transport and its GH releasing effect is linked to the presence of calcium.

Role of hypothalamic factors (growth-hormone-releasing hormone and gamma-aminobutyric acid) in the regulation of growth hormone secretion in the neonatal and adult rat.

N-methyl aspartic acid (NMA) was without effect on the pituitary growth hormone (GH) secretion of adult and neonatal rats in vitro. Administration of NMA resulted in a rapid rise of plasma GH levels in intact but not in arcuate-nucleus-lesioned adult rats, indicating that NMA stimulated GH-releasing hormone (GRH) secretion. In 2-day-old rats, both NMA and gamma-aminobutyric acid (GABA) elevated plasma GH levels in a dose-related fashion; GRH administration was without effect. The elevation of plasma GH levels after NMA injection was reduced by administering an antibody to GRH. These results indicate that GH secretion is partly regulated by endogenous GRH in the newborn rat but that the elevation of plasma GH levels after GABA is not mediated by GRH. The high plasma GH levels seen in the newborn rat may result from the independent action of GABA and GRH but the effect of other factors cannot be excluded either.

Rat milk stimulates pituitary growth hormone secretion of neonatal pituitaries in vitro.

Aqueous extracts of rat milk stimulated growth hormone (GH) secretion from superfused pituitaries of two-day old rats. The GH stimulatory effect of milk increased with the time elapsed postpartum; growth hormone releasing hormone and thyrotropin releasing hormone seem to be the major milk borne GH releasing factors. These results indicate that milk intake may play a role in maintaining the high plasma GH levels observed in the neonatal period.

gamma-Aminobutyric acid stimulates pituitary growth hormone secretion in the neonatal rat. A superfusion study.

The putative inhibitory neurotransmitter gamma-aminobutyric acid (GABA) elicited a dose-dependent increase in GH secretion from the pituitary of newborn rats. GH secretion increased within 3 min after GABA administration with a peak response at 5-6 min. The lowest effective dose of the GABA agonist muscimol was about 10 times smaller than that of GABA. The GABA effect was antagonized by picrotoxin and bicuculline, suggesting that GABA acts at GABA-A type receptors. The pituitary responsiveness to GABA gradually decreased during the second and third postnatal weeks. If the neonatal pituitaries were continuously exposed to GABA for 3 h GH secretion rapidly increased to a maximum within the first 10 min and then gradually decreased to a less elevated level by 1 h and remained at this level for the next 2 h. After 3 h of GABA exposure muscimol had no effect on GH secretion but human pancreatic GH-releasing factor stimulated it, indicating receptor desensitization during prolonged GABA administration. The significance of GABAergic regulation of GH secretion in the neonate is emphasized by the finding that simultaneous administration of picrotoxin diminished the GH releasing activity of the hypothalamic extract of 2-day-old rats by more than 60%. These results indicate that in the postnatal period the regulation of GH secretion differs from that of the adult animal and GABA might play an important role in the maintenance of the high GH secretion during the first days of life.

Vasopressin secretion as a possible target of the gamma-aminobutyric acid-mediated component of the corticosteroid feedback effect.

The inhibition of gamma-aminobutyric acid (GABA) synthesis did not interfere with the suppressive effect of dexamethasone on the stress-induced rise of plasma corticosterone levels in vasopressin-deficient homozygous Brattleboro rats. In dexamethasone-treated heterozygous rats corticosterone and vasopressin secretion increased after stress provided GABA synthesis was inhibited. The results indicate that inhibition of corticotrophin secretion by corticosteroids may in part be mediated by enhancement of GABA synthesis and a consequent inhibition of vasopressin release.