Different expression of the two dopaminergic D2 receptors, D2415 and D2444, in two types of lactotroph each characterised by their response to dopamine, and modification of expression by sex steroids.

Dopamine inhibits prolactin liberation acting via the D2 type receptor. Two different electrophysiological responses to dopamine have been shown to characterise two types of lactotroph isolated from the lactating female rat. It is now known that differential splicing of the pre-messenger RNA coding for the D2 receptor leads to the production of two D2 subtypes, D2(415) and D2(444). These subtypes differ in the region which is believed to be responsible for the binding of G proteins, and could thus lead to the activation of different intracellular second messenger systems. Here we show that the pre-messenger RNA for the D2 receptor is differentially spliced in such a way that the ratio D2(415)/D2(444) is significantly different (2.91 +/- 0.6 vs 1.29 +/- 0.14) between two populations of lactotrophs, each enriched in cells showing one type of response to DA. We further show that the ratio D2(415)/D2(444) can be changed by treatment of prolactin cells in primary culture with progesterone or testosterone. Estrogen did not change the ratio, but diminished the total amount of D2 cDNA. Regulation of differential splicing by sex steroids could provide a mechanism for modifying lactotroph responsiveness to DA in different physiological situations.

Characterization of interleukin-1 receptor antagonist isoform expression in the brain of lipopolysaccharide-treated rats.

The endogenous interleukin-1 receptor antagonist is the natural inhibitor of the biological effects of interleukin-1 during inflammation. Interleukin-1 receptor antagonist refers to three isoforms: one secreted and two intracellular forms (types I and II). The objective of the present study was to investigate the expression of interleukin-1 receptor antagonist isoforms in the rat brain in vivo in response to an i.p. injection of lipopolysaccharide. The interleukin-1 receptor antagonist was studied at the messenger and protein levels by reverse transcription-polymerase chain reaction and western blot analysis, respectively. Interleukin-1 receptor antagonist messenger RNA was constitutively expressed in the brain and its expression increased in response to lipopolysaccharide. The three interleukin-1 receptor antagonist protein isoforms were up-regulated after lipopolysaccharide treatment in a time-dependent manner. Their relative expression differed according to the isoform and brain region studied. Double immunofluorescence staining revealed interleukin-1 receptor antagonist positive neurons and microglia in hippocampus 24h after lipopolysaccharide stimulation. These results demonstrate for the first time that brain cells are able to produce interleukin-1 receptor antagonist isoforms in response to a peripheral immune challenge with a predominance of the secreted over intracellular forms.

Identification of epidermal growth factor-secreting cells in the anterior pituitary of lactating female rats.

Epidermal growth factor (EGF) is synthesized and secreted by mammalian anterior pituitary cells. It stimulates GH and prolactin (PRL) secretion, but the cellular origin of EGF is relatively unexplored. The objective of this study was to characterize the cells that secrete EGF in the anterior pituitary of lactating rats. An EGF reverse haemolytic plaque assay (RHPA) was used to identify EGF-secreting cells and this RHPA was combined with immunofluorescence using antibodies to the six major adenohypophysial hormones (i.e. PRL, GH, LH, FSH, TSH and ACTH). Approximately 20% (20.33 +/- 2.96%) of the cells in the pituitary of lactating rats secrete EGF. The EGF-secreting cell population was composed of the following labelled cells: PRL (27%), GH (20%), LH (18%), FSH (14%), TSH (14%) and ACTH (5%). The present study showed that EGF is released by a subpopulation of anterior pituitary cells composed of all the classic hormone-containing cells.

In vitro Effects of 17Beta-Estradiol on Thyrotropin-Releasing Hormone-Induced and Dopamine-lnhibited Prolactin Release from Adult Male Rat Lactotrophs in Primary Culture.

Abstract Continuous cell perifusion and reverse hemolytic plaque assay have been used to show a regulatory action of 17 beta-estradiol on lactotroph responsiveness to thyrotropin-releasing hormone (TRH) or dopamine (DA) in vitro. Lactotroph-enriched cell cultures were obtained from adult male rats after trypsinization and mechanical dissociation followed by separation on a continuous bovine serum albumin gradient at unit gravity. After 7 days in culture, perifusion experiments showed that prolactin was continuously released and this release was increased by TRH and decreased by DA. Both TRH-induced secretion and DA-induced inhibition of prolactin release were dose-dependent with a half maximal effect obtained at 7 x 10(-9) M for TRH and at 10(-9) M for DA. It was shown by reverse hemolytic plaque assay that about 55% of the cells were plaque-forming (lysis of red blood cells) and were thus identified as prolactin-secreting cells. This was similar to a previous result obtained by immunofluorescent staining. Heterogeneity among lactotrophs with regard to the quantity of prolactin released was clearly shown by the varying plaque areas in all preparations. In order to make a quantitative analysis of the effect of 17 beta-estradiol on TRH-stimulation and DAergic inhibition in these heterogeneous prolactin cells, they were divided into two groups: large plaques (>/= 3 x 10(3)mu m(2)) constituted about 35% of all plaque-forming cells, and small plaques (< 3 x 10(3)mu m(2)), about 65%. Pretreatment with 17beta-estradiol (10(-8) M) either for 10 h or 48 h markedly increased TRH-stimulated prolactin release and decreased the inhibitory effect of DA both in perifusion and reverse hemolytic plaque assay experiments. However, these pretreatments did not change the values of half maximum dose for TRH and DA. TRH transformed about 7% of the small plaques into large plaques and this proportion was increased to 25% after 17beta-estradiol treatment. On the contrary, DA and its more stable analogue bromocriptine increased the percentage of small plaques by 10% to 15% but this effect was decreased after 17beta-estradiol treatment. We conclude that: 1) Normal rat pituitary lactotrophs show heterogeneity with respect to their spontaneous release and responsiveness to TRH and DA; 2) pretreatment with 17beta-estradiol increases the response to TRH and decreases the response to DA without altering the doses at which they have half maximal effect; 3) there is no significant difference between the effect of 17beta-estradiol obtained after 10 h and after 48 h pretreatment.

Apoptosis occurs independently of the release of interleukin-1 beta in the anterior pituitary of end-lactating rats.

The mechanism of Interleukin-1 beta (IL-1 beta) release remains unknown. Because of the absence of typical peptide signal on the precursor, IL-1 beta is not secreted through the classical pathway. The aim of this study was to determine whether IL-1 beta is released during apoptosis, as has been reported for activated macrophages. We chose anterior pituitary cells of end-lactating rats because of their capacity to produce IL-1 beta spontaneously and because this organ undergoes cellular degeneration. The combination of two techniques, reverse haemolytic plaque assay (RHPA) and terminal transferase dUTP nick end labelling (TUNEL), allowed us to observe simultaneously the release of IL-1 beta and apoptosis. Our results show that in these conditions apoptosis is not the mechanism of IL-1 beta release.

Evidence for a relationship between lactotroph heterogeneity and physiological context.

Two lactotroph subpopulations differing in their functional properties have previously been isolated from lactating female rats. It was found that adult female rats at proestrus similarly yield two subpopulations of lactotrophs, whereas those from adult female rats at metestrus show different properties and in males only one functional population is found. As the period of lactation and proestrus are characterised by high circulating estrogen levels, estrogen is considered to modify lactotroph properties in the female, and as the functional differences concern the lactotroph responsiveness to TRH, it is supposed that lactotroph heterogeneity provides a means for particular secretory patterns during lactation and at proestrus.

Production of interleukin-1 receptor antagonist isoforms by microglia in mixed rat glial cells stimulated by lipopolysaccharide.

Although the natural interleukin-1 receptor antagonist (IL-1Ra) has been shown to be produced by microglial cells in response to immune stimuli, nothing was known about the ability of these cells in primary culture to produce the different isoforms of IL-1Ra. Using RT-PCR, we first confirmed that mixed glial cell cultures from newborn rats respond to the cytokine inducer, lipopolysaccharide, by synthesizing IL-1Ra mRNA. Using double immunostaining, we showed that IL-1Ra was detected in microglia but not in astrocytes. Using Western blotting, we finally demonstrated that the IL-1Ra1 isoform was secreted in the supernatant of mixed glial cell cultures, and its production increased in response to lipopolysaccharide. The three different IL-1Ra isoforms were constitutively expressed in cell lysates and their levels increased after lipopolysaccharide treatment, except for IL-1Ra3. These results point to the ability of microglial cells in primary culture to produce the different isoforms of IL-1Ra.

EGF release by rat gonadotroph cells: characteristics and effects of LHRH.

The ability of anterior pituitary cells of immature female rats to secrete epidermal growth factor (EGF) was studied using the reverse hemolytic plaque assay. An average of 22% of cells (22.12 +/- 0.49%) spontaneously released low amount of EGF, as measured by the small area of plaques surrounding cells (1230 +/- 67 micron2). To determine the cellular origin of EGF from the rat anterior pituitary gland, a combination of immunocytochemistry and reverse hemolytic plaque assay was used. Most of the EGF-secreting cells were identified as luteinizing hormone (LH) containing cells (72.14 +/- 2.97%). Treatment with luteinizing hormone releasing hormone (LHRH; 10 nM) significantly increased the plaque area formed around EGF-secreting cells (2438 +/- 114 micron2) without altering the number of EGF-secreting cells. These results indicate that EGF is mostly secreted by gonadotrophs and that this EGF release is enhanced by LHRH.

Reproducibility of 1H-NMR integrals: a collaborative study.

The quantitative use of NMR spectroscopy was investigated by a reproducibility study of 1H-NMR integrals involving five laboratories. A significant laboratory effect was found confirming the difficulty to obtain very precise data by integration of complex signals. The reproducibility of any NMR assay measurement, which requires a high precision should be validated by an interlaboratory study.

Human-associated Staphylococcus aureus strains within great ape populations in Central Africa (Gabon).

The risk of serious infections caused by Staphylococcus aureus is well-known. However, most studies regarding the distribution of (clinically relevant) S. aureus among humans and animals took place in the western hemisphere and only limited data are available from (Central) Africa. In this context, recent studies focused on S. aureus strains in humans and primates, but the question of whether humans and monkeys share related S. aureus strains or may interchange strains remained largely unsolved. In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Therefore, a primate facility at the International Centre for Medical Research of Franceville (Gabon) was screened. We detected among the primates a common human S. aureus strain, belonging to the spa-type t148. It was isolated from three different individuals of the western lowland gorilla (Gorilla gorilla gorilla), of which one individual showed a large necrotizing wound. This animal died, most probably of a staphylococcal sepsis. Additionally, we discovered the t148 type among chimpanzees (Pan troglodytes) that were settled in the immediate neighbourhood of the infected gorillas. A detailed analysis by pulsed field gel electrophoresis showed that the gorilla and chimpanzee isolates represented two closely related strains. To our knowledge, this is the first report of a human-associated S. aureus strain causing disease in great apes. The simultaneous detection in gorillas and chimpanzees indicated an interspecies transmission of this S. aureus strain. Our results recommend that protection of wild animals must not only be based on habitat conservation, but also on the assessment of the risk of contact with human pathogens.

The physiological and behavioural development of diving in Australian fur seal (Arctocephalus pusillus doriferus) pups.

The physiological and behavioural development of diving was examined in Australian fur seal (Arctocephalus pusillus doriferus) pups to assess whether animals at weaning are capable of exploiting the same resources as adult females. Haematocrit, haemoglobin and myoglobin contents all increased throughout pup development though total body oxygen stores reached only 71% of adult female levels just prior to weaning. Oxygen storage components, however, did not develop at the same pace. Whereas blood oxygen stores had reached adult female levels by 9 months of age, muscle oxygen stores were slower to develop, reaching only 23% of adult levels by this age. Increases in diving behaviour corresponded to the physiological changes observed. Pups spent little time (<8%) in the water prior to moulting (age 1-2 months) whereas following the moult, they spent >27% of time in the water and made mid-water dives (maximum depth 35.7 +/- 2.9 m) with durations of 0.35 +/- 0.03 min. By 9 months (just prior to weaning), 30.5 +/- 9.3% of all dives performed were U-shaped benthic dives (maximum depth 65.0 +/- 6.0 m) with mean durations of 0.87 +/- 0.25 min, significantly shorter than those of adult females. These results suggest that while Australian fur seal pups approaching the age of weaning are able to reach similar depths as adult females, they do not have the physiological capacity to remain at these depths for sufficient durations to exploit them to the same efficiency.

The enzyme indoleamine 2,3-dioxygenase is induced in the mouse brain in response to peripheral administration of lipopolysaccharide and superantigen.

The essential amino-acid, L-tryptophan, is the precursor of serotonin. Its availability in the brain is controlled by indoleamine 2,3-dioxygenase (IDO). This enzyme is inducible by cytokines such as interferon-gamma (IFN-gamma) and is the first and rate-limiting enzyme of the catabolism pathway of tryptophan. Since induction of IDO has been proposed to mediate the influence of cytokines on mood in patients with various somatic disorders, the present study aimed at analyzing the relationships between changes in brain IDO activity and serum IFN-gamma levels in response to peripheral immune stimulation by lipopolysaccharide (LPS) and superantigen in mice. Each of these treatments induced an increase in serum IFN-gamma at 6 h post-treatment followed 24 h later by a two-fold increase in IDO activity in the brain. These results support the involvement of peripheral IFN-gamma in the control of L-tryptophan catabolism in the brain.

Rat microglial cells secrete predominantly the precursor of interleukin-1beta in response to lipopolysaccharide.

Little is known on the forms of interleukin-1beta (IL-1beta) that are produced by microglial cells in the nervous system. Mixed glial cell cultures of rats produced IL-1beta in response to lipopolysaccharide (LPS). Using Western blot, pro-IL-1beta was found to be localized both intracellularly and in the supernatant, whereas mature IL-1beta was found only in the supernatant but in lower quantities than pro-IL-1beta. Immunocytochemistry confirmed that microglial cells are the exclusive source of IL-1beta. Blockade of the IL-1beta-converting enzyme (ICE) by Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO) decreased the levels of mature IL-1beta but had no effect on pro-IL-1beta. Release of pro-IL-1beta was not associated with cell death nor with the extracellular release of ICE. Using gelatin zymography, glial cells were found to express constitutive matrix metalloproteinases (MMP) in the form of MMP-2. Exposure to LPS induced MMP-9 expression in a time-dependent manner similar to the pro-IL-1beta expression profile. MMP activation and inhibition experiments indicated a possible role of MMPs in the cleavage of pro-IL-1beta but not in the generation of mature IL-1beta. Microglial cells share with macrophages the ability to release large amounts of pro-IL-1beta of which the extracellular role remains to be determined.

Assessing a relationship between bone microstructure and growth rate: a fluorescent labelling study in the king penguin chick (Aptenodytes patagonicus).

Microstructure-function relationships remain poorly understood in primary bone tissues. The relationship between bone growth rate and bone tissue type, although documented in some species by previous works, remains somewhat unclear and controversial. We assessed this relationship in a species with extreme adaptations, the king penguin (Aptenodytes patagonicus). These birds have a peculiar growth, interrupted 3 months after hatching by the austral winter. Before this interruption, chicks undergo extremely rapid statural and ponderal growth. We recorded experimentally (by means of fluorescent labelling) the growth rate of bone tissue in four long bones (humerus, radius, femur and tibiotarsus) of four king penguin chicks during their fastest phase of growth (3-5 weeks after hatching) and identified the associated bone tissue types ('laminar', 'longitudinal', 'reticular' or 'radial' fibro-lamellar bone tissue). We found the highest bone tissue growth rate known to date, up to 171 microm day(-1) (mean 55 microm day(-1)). There was a highly significant relationship between bone tissue type and growth rate (P<10(-6)). Highest rates were obtained with the radial microarchitecture of fibro-lamellar bone, where cavities in the woven network are aligned radially. This result supports the heuristic value of a relationship between growth rate and bone primary microstructure. However, we also found that growth rates of bone tissue types vary according to the long bone considered (P<10(-5)) (e.g. growth rates were 38% lower in the radius than in the other long bones), a result that puts some restriction on the applicability of absolute growth rate values (e.g. to fossil species). The biomechanical disadvantages of accelerated bone growth are discussed in relation to the locomotor behaviour of the chicks during their first month of life.

Angiotensin II stimulates prolactin release in the rhesus monkey.

The effect of angiotensin II (AII) on plasma prolactin (PRL) concentrations was evaluated in ovariectomized rhesus monkeys which had been conditioned to chair restraining. Intracerebro-ventricular (i.v.t.) microinjections of AII increased PRL plasma levels in a dose-dependent manner. PRL values peaked at 10 min after injection and declined rapidly thereafter. Peak levels of PRL after 50 micrograms AII were approximately 7 times higher than those of baseline controls and were significantly reduced when 500 micrograms saralasin were injected via the ventricular route 15 min before AII injections. Measurements of vasopressin (AVP) plasma levels in some experiments showed that AVP release occurred following any dose of AII, irrespective of PRL liberation. In 2 animals, central injections of AII induced a dose-related increase in blood pressure. This study shows that, in primates, AII is able to influence PRL secretion via specific AII receptors. This action of AII is independent of AVP release and may be indirectly mediated through vasoactive effects of the octapeptide.

Evidence that TRH controls prolactin release from rat lactotrophs by stimulating a calcium influx.

Prolactin (PRL) release and intracellular free calcium concentration [Ca2+]i were measured in two populations of normal rat lactotrophs (light and heavy fractions) in culture. Spontaneous PRL release of heavy fraction cells was more sensitive to dihydropyridines (DHPs; Bay K 8644 and nifedipine) when compared to the light fraction lactotrophs. The stimulatory effect of thyrotropin-releasing hormone (TRH) on PRL release from heavy fraction cells was inhibited by Cd2+ and mimicked by Bay K 8644. Indo-1 experiments revealed that TRH-increased [Ca2+]i was reversibly inhibited by Cd2+. In a Ca(2+)-free EGTA-containing medium, TRH did not modify [Ca2+]i.

[Histamine and prolactin liberation in the rhesus monkey]. / Histamine et libération de prolactine chez le singe rhesus.

The effect of intra venous (i.v.) or intra cerebroventriculaire (i.c.v.) administration of histamine (HA) on plasma prolactin (PRL) levels was investigated in ovariectomized Rhesus Monkeys. Intra venous injection of 50 micrograms/kg HA increased the plasma PRL concentration but icv administration of 10 and 50 micrograms decreased PRL plasma levels. Intra venous injection of 2-thiazolyl-éthylamine, a H1 receptor agonist, rapidly stimulated PRL release (peak PRL concentration at 5 min) suggesting a direct effect on the pituitary. In contrast intra venous administration of the H2 receptor agonist, impromidine, inhibited PRL release at low doses. High doses of impromidine increased PRL concentrations but this effect was delayed (PRL peak values were reached at 20 minutes). Our results show that HA may influence PRL release in the primate via H1 and H2 receptors located at both pituitary and central levels.

Lipopolysaccharide induces sickness behaviour in rats by a vagal mediated mechanism.

To assess the possibility that lipopolysaccharide (LPS) induces sickness behaviour by activating primary afferent nerves, the effects of LPS (1.25 mg kg-1, intraperitoneally) were compared in vagotomized and sham-operated rats. Subdiaphragmatic vagotomy blocked the LPS-induced depression of social investigation but had no effect on LPS-induced increases in levels of IL-1 beta in plasma and peritoneal macrophages and on LPS-induced changes in dehydrogenase activity of peritoneal macrophages.