Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups.METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method.RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol).CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Impaired beta-adrenergic response and decreased L-type calcium current of hypertrophied left ventricular myocytes in postinfarction heart failure

Infarct-induced heart failure is usually associated with cardiac hypertrophy and decreased ß-adrenergic responsiveness. However, conflicting results have been reported concerning the density of L-type calcium current (I Ca(L)), and the mechanisms underlying the decreased ß-adrenergic inotropic response. We determined I Ca(L) density, cytoplasmic calcium ([Ca2+]i) transients, and the effects of ß-adrenergic stimulation (isoproterenol) in a model of postinfarction heart failure in rats. Left ventricular myocytes were obtained by enzymatic digestion 8-10 weeks after infarction. Electrophysiological recordings were obtained using the patch-clamp technique. [Ca2+]i transients were investigated via fura-2 fluorescence. ß-Adrenergic receptor density was determined by [³H]-dihydroalprenolol binding to left ventricle homogenates. Postinfarction myocytes showed a significant 25% reduction in mean I Ca(L) density (5.7 ± 0.28 vs 7.6 ± 0.32 pA/pF) and a 19% reduction in mean peak [Ca2+]i transients (0.13 ± 0.007 vs 0.16 ± 0.009) compared to sham myocytes. The isoproterenol-stimulated increase in I Ca(L) was significantly smaller in postinfarction myocytes (Emax: 63.6 ± 4.3 vs 123.3 ± 0.9% in sham myocytes), but EC50 was not altered. The isoproterenol-stimulated peak amplitude of [Ca2+]i transients was also blunted in postinfarction myocytes. Adenylate cyclase activation through forskolin produced similar I Ca(L) increases in both groups. ß-Adrenergic receptor density was significantly reduced in homogenates from infarcted hearts (Bmax: 93.89 ± 20.22 vs 271.5 ± 31.43 fmol/mg protein in sham myocytes), while Kd values were similar. We conclude that postinfarction myocytes from large infarcts display reduced I Ca(L) density and peak [Ca2+]i transients. The response to ß-adrenergic stimulation was also reduced and was probably related to ß-adrenergic receptor down-regulation and not to changes in adenylate cyclase activity.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Stimulatory effects of adenosine on prolactin secretion in the pituitary gland of the rat

We investigated the effects of adenosine on prolactin (PRL) secretion from rat anterior pituitaries incubated in vitro. The administration of 5-N-methylcarboxamidoadenosine (MECA), an analog agonist that preferentially activates A2 receptors, induced a dose-dependent (1 nM to 1 µM) increase in the levels of PRL released, an effect abolished by 1,3-dipropyl-7-methylxanthine, an antagonist of A2 adenosine receptors. In addition, the basal levels of PRL secretion were decreased by the blockade of cyclooxygenase or lipoxygenase pathways, with indomethacin and nordihydroguaiaretic acid (NDGA), respectively. The stimulatory effects of MECA on PRL secretion persisted even after the addition of indomethacin, but not of NDGA, to the medium. MECA was unable to stimulate PRL secretion in the presence of dopamine, the strongest inhibitor of PRL release that works by inducing a decrease in adenylyl cyclase activity. Furthermore, the addition of adenosine (10 nM) mimicked the effects of MECA on PRL secretion, an effect that persisted regardless of the presence of LiCl (5 mM). The basal secretion of PRL was significatively reduced by LiCl, and restored by the concomitant addition of both LiCl and myo-inositol. These results indicate that PRL secretion is under a multifactorial regulatory mechanism, with the participation of different enzymes, including adenylyl cyclase, inositol-1-phosphatase, cyclooxygenase, and lipoxygenase. However, the increase in PRL secretion observed in the lactotroph in response to A2 adenosine receptor activation probably was mediated by mechanisms involving regulation of adenylyl cyclase, independent of membrane phosphoinositide synthesis or cyclooxygenase activity and partially dependent on lipoxygenase arachidonic acid-derived substances.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

In vitro cell activating properties of the composite ribosomal vaccine D53

D53 (RibomuntyR) is a composite vaccine made of immunogenic ribosomes from 4 bacterial species (Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and Streptococcus pneumoniae) associated with a membrane proteoglycan from a non encapsulated strain of Klebsiella pneumoniae. D53 is a potent inducer of interleukin-1 production by mouse BALB/c spleen cells as shown by the C3H/HeJ thymocyte co-stimulation assay. Furthermore D53 triggers DNA synthesis by mouse spleen cells and induces the maturation of B lymphocytes into immunoglobulin secreting cells. Polyclonal B cell activation by D53 was readily achieved in the C3H/HeJ strain which is deficient in its response to E. coli lipopolysaccharide. The proliferative response to D53 was abrogated by removal of B cells from the spleen cell suspension, but it was not altered after depletion of T cells or adherent cells. D53 induced polyclonal B cell activation of spleen cells from athymic nude mice and from CBA/N mice. Each component of D53 induced polyclona B cell activation except ribosomes from Streptococcus pneumoniae. Each triggered Interleukin-1 synthesis except ribosomes from Klebsiella penumoniae. These in vitro properties may account for some of the in vivo immunostimulating properties of this composite vaccine.

Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating Protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

Regulating inflammation through the anti-inflammatory enzyme platelet-activating factor-acetylhydrolase

Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.

Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

Chagas disease (CD) causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF) causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs), which is the main characteristic of the G protein-coupled receptors (GPCRs), including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1%, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

Hordein polypeptide patterns in relation to malting quality in Brazilian barley varieties

Since there is evidence that malting quality is related to the storage protein (hordein) fraction, in the present work the hordein polypeptide patterns from 13 barley (Hordeum vulgare L.) varieties of different malting quality were analysed in order to explore the feasibility of using hordein electrophoresis to assist in the selection of malting barleys. The formation of clusters separating the varieties with higher malting quality from the others with lower quality suggests that there is a relationship between the general hordein polypeptide pattern and malting quality in the varieties analysed. By the Sperman's correlation test three hordein bands correlated negatively with malting quality in the germplasm studied.

Hypothalamic-pituitary-gonadal function in men with liver cirrhosis before and after liver transplantation

Objective: To evaluate the influence of end-stage liver disease and orthotopic liver transplantation in the pituitary function and hormone metabolism before and after liver transplantation.Methods: In a prospective study, serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and prolactin (PRL) of 30 male patients with cirrhosis were determined two to four hours before and six months after liver transplantation. The results were compared according to the Model for End-stage Liver Disease (MELD).Results: male patients with liver cirrhosis have hypogonadism. FSH was normal, but inappropriately low due to androgen failure; E2 and PRL, on their turn, were high. After liver transplantation, FSH and LH levels increased (p < 0.05), whereas E2 and PRL normalized (p < 0.05). The MELD score did not influence changes in FSH, PRL and LH, however, the more severe the cirrhosis was, the more significant was the normalization of E2 (p = 0.01).Conclusion: Patients with cirrhosis and male hypogonadism have inappropriately normal levels of FSH and LH, associated with an increase in E2 and LRP. After liver transplantation, FSH and LH increased, while E2 and PRL returned to normal. Changes in E2 levels were most pronounced in patients with MELD > 18. The severity of cirrhosis had no influence on FSH, PRL and LH.

Effects of bromocriptine on serum prolactin levels, pituitary weight and immunoreactive prolactin cells in estradiol-treated ovariectomized rats: an experimental model of estrogen-dependent hyperprolactinemia

The present study was designed to assess the effects of bromocriptine, a dopamine agonist, on pituitary wet weight, number of immunoreactive prolactin cells and serum prolactin concentrations in estradiol-treated rats. Ovariectomized Wistar rats were injected subcutaneously with sunflower oil vehicle or estradiol valerate (50 or 300 µg rat-1 week-1) for 2, 4 or 10 weeks. Bromocriptine (0.2 or 0.6 mg rat-1 day-1) was injected daily during the last 5 or 12 days of estrogen treatment. Data were compared with those obtained for intact control rats. Administration of both doses of estrogen increased serum prolactin levels. No difference in the number of prolactin cells in rats treated with 50 µg estradiol valerate was observed compared to intact adult animals. In contrast, rats treated with 300 µg estradiol valerate showed a significant increase in the number of prolactin cells (P<0.05). Therefore, the increase in serum prolactin levels observed in rats treated with 50 µg estradiol valerate, in the absence of morphological changes in the pituitary cells, suggests a "functional" estrogen-induced hyperprolactinemia. Bromocriptine decreased prolactin levels in all estrogen-treated rats. The administration of this drug to rats previously treated with 300 µg estradiol valerate also resulted in a significant decrease in pituitary weight and number of prolactin cells when compared to the group treated with estradiol alone. The general antiprolactinemic and antiproliferative pituitary effects of bromocriptine treatment reported here validate the experimental model of estrogen-induced hyperprolactinemic rats

Pituitary-thyroid axis in short- and long-term experimental diabetes mellitus

Short-term experimental diabetes mellitus (DM) produces a significant decrease in serum thyroid hormones, a decreased or normal serum thyroid-stimulating hormone (TSH) and a reduction in hepatic and renal T4-5'-deiodination. However, little is known about the effects of chronic diabetes mellitus on the pituitary-thyroid axis function. We evaluated the changes induced by very short-term (6 days), short-term (15 days) and chronic (6 months) streptozotocin-induced diabetes mellitus in 3-month old female Dutch-Miranda rat serum T4, serum TSH and T4-5'-deiodinase activity in the thyroid and pituitary glands. Serum hormones were determined by specific radioimmunoassays. Iodothyronine-5'-deiodinase activities were assayed in the thyroid and pituitary microsomal fractions using 2 µM T4 as substrate. Mean serum T4 was significantly decreased from 3.3 to 2.0 µg/dl 6 days after diabetes mellitus induction, and from 2.2 to 1.5 µg/dl after 15 days of DM, with no significant changes in serum TSH, indicating a decreased pituitary TSH responsiveness to the diminished suppression by T4, even though pituitary T4-5'-deiodinase activity was unchanged. Thyroid T4-5'-deiodinase was unchanged after 6 days of diabetes mellitus, but was significantly increased from 20.6 to 37.0 pmol T3/mg protein after 15 days. Six months after diabetes mellitus induction, both serum T4 and thyroid T4-5'-deiodinase returned to normal ranges and serum TSH was unchanged, although pituitary T4-5'-deiodinase was now significantly decreased from 2.7 to 1.7 pmol T3/mg protein. These findings indicate that some kind of adaptation to chronic insulinopenia may occur at the thyroid level, but this does not seem to be true for the pituitary

Release of plasma atrial natriuretic peptide after volume expansion is not related to pituitary-adrenal axis diurnal variation in normal subjects

The existence of a circadian rhythm of atrial natriuretic peptide (ANP) in humans is controversial. We studied the plasma ANP response to isotonic blood volume expansion in the morning and in the afternoon and its relationship with adrenocorticotropic hormone (ACTH)-cortisol diurnal variation in seven normal subjects. Basal plasma ANP level was similar in the morning (19.6 ± 2.4 pg/ml) and in the afternoon (21.8 ± 4.8 pg/ml). The ANP peak obtained with saline infusion (0.9% NaCl, 12 ml/kg) in the morning (49.4 ± 8 pg/ml) did not differ from that obtained in the afternoon (60.3 ± 10.1 pg/ml). There was no correlation between the individual mean cortisol and ACTH levels and the ANP peak obtained with saline infusion. These data indicate no diurnal variation in plasma ANP secretion induced by blood volume expansion and no relationship between plasma ANP peak and ACTH-cortisol diurnal variation