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A I Martín, E Castillero, M Granado, M López-Menduiña, M A Villanúa and A López-Calderón

Adjuvant-induced arthritis is a model of rheumatoid arthritis that induces cachexia. In other cachectic situations, there is an increase in lipolysis resulting in a loss of adipose tissue mass. The aim of this work was to analyse the effect of chronic arthritis, induced by adjuvant injection, on white adipose tissue (WAT). For this purpose, rats were killed 10 days after adjuvant injection, when the first external symptoms appeared, on days 15 and 22 when the external signs of the illness reach their severest level. As arthritis decreases food intake, a pair-fed group was also included. Serum concentrations of insulin, leptin, adiponectin, glycerol and nitrites, as well as gene expression of leptin, adiponectin, hormone-sensitive lipase (HSL), fatty acid synthase (FAS), tumour necrosis factor α and zinc-α2-glycoprotein (ZAG) were determined. Arthritis decreased food intake between days 5 and 16, but not during the last 5 days of the experiment. There was a marked decrease in relative adipose tissue weight and in serum leptin and adiponectin as well as in their gene expression in WAT in arthritic rats. Arthritis decreased the gene expression of FAS in the WAT. However, none of these effects was found in pair-fed rats. Arthritis did not increase lipolysis, since arthritic rats have lower serum concentrations of glycerol, HSL mRNA in WAT, as well as liver ZAG mRNA than the pair-fed or control rats. These data suggest that in chronic arthritis the decrease in white adipose mass is secondary to a reduced adipose lipogenesis, and this effect is not mainly due to the decrease in food intake.

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A I Martín, M López-Menduiña, E Castillero, M Granado, M A Villanúa and A López-Calderón

The aim of this work was to analyse the role of cyclooxygenase-2 (Ptgs2) in endotoxin-induced decrease in Igf1 and Igf binding protein-3 (Igfbp3). For this purpose, male Wistar rats were injected with lipolysaccharide (LPS) and/or the Ptgs2 inhibitor meloxicam. LPS induced a significant decrease (P<0.01) in serum concentrations of Igf1 and Igfbp3 and their mRNAs in the liver. Meloxicam administration prevented the inhibitory effect of LPS injection on serum Igf1 and its liver mRNA. By contrast, meloxicam administration was unable to modify the inhibitory effect of LPS on Igfbp3. LPS injection also induced a decrease in GH receptor (Ghr) mRNA in the liver, and meloxicam attenuated this effect. In order to elucidate a direct action of the Ptgs2 inhibitor on the liver cells, the effect of LPS and/or meloxicam was studied in primary cultures of hepatocytes with non-parenchymal cells. LPS decreased Igf1 and Ghr but not Igfbp3 gene expression in liver cells in culture. Meloxicam administration attenuated the inhibitory effect of LPS on Igf1 mRNA, whereas it did not modify the decrease in Ghr mRNA after LPS. The effect of meloxicam on the LPS response does not seem to be mediated by changes in nitric oxide or tumour necrosis factor (Tnf) production, since meloxicam did not modify the stimulatory effect of LPS on nitric oxide or Tnfα gene expression both in vivo and in vitro. All these data suggest that LPS-induced Ptgs2 activation decreases Igf1 gene expression in liver cells.

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A. López Bernal, Anne B. M. Anderson and A. C. Turnbull

Cortisol: cortisone interconversion was studied in human decidua obtained from three groups of patients at term (37-42 weeks): before the onset of labour (at elective Caesarean section), after labour of spontaneous onset, and after labour of induced onset. When intact tissue was incubated with [3H]cortisol or [3H]cortisone in phosphate buffer without added substrate or cofactors, cortisone to cortisol was the dominant conversion. However, when damaged cells or tissue homogenates were used in the same conditions, the dominant direction of the reaction was reversed, with a large increase in oxidative (cortisol to cortisone) activity. Cortisol: cortisone interconversion was similar in the three groups of samples using either intact tissue or homogenates, as was the total 11β-hydroxysteroid dehydrogenase (11β-HSD) activity measured in tissue homogenates in the presence of added substrate (cortisol or cortisone) and cofactors (NADP+ or NADPH). Endogenous cortisol concentrations in decidua were higher than those of cortisone, and the ratio of cortisol to cortisone was similar in the three groups. These findings suggest that there are no changes in human decidual 11β-HSD activity in relation to parturition. Specific activity of 11β-HSD decreased at high protein concentrations, suggesting the presence of some enzyme inhibitor(s) in homogenized decidual tissue.

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A I Martín, J Fernández-Ruiz and A López-Calderón

Abstract

Acute stress is known to increase LH secretion and the release of central norepinephrine (NE) in intact rats. Studies were performed to analyse the role of catecholamines in acute stress-induced LH release in male rats. Injection of α-methyl-p-tyrosine (αMPT) and diethyldithiocarbamate (DDC), catecholamine synthesis inhibitors, significantly decreased both hypothalamic concentration of NE and serum LH. Restraint for 30 min evoked an increase in serum LH in saline-treated rats, whereas αMPT and DDC administration blocked the stress-induced LH release. The effects of α1-, α2- and β-adrenoreceptor antagonists on the LH response to restraint stress were also studied. Propranolol treatment did not modify serum LH in either unstressed or stressed rats. The two α-adrenergic receptor antagonists prazosin and yohimbine prevented the restraint-induced LH release; however, prazosin but not yohimbine significantly decreased the serum concentration of LH in unstressed rats. These data suggest that the acute stress-induced increase in LH secretion is mediated through the activation of α2-adrenergic receptors.

Journal of Endocrinology (1995) 144, 511–515

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M. López, M. A. Castrillón and A. N. Tchernitchin

ABSTRACT

Oestrogen induces a migration of eosinophil leukocytes to the uterus where, it is suggested, these cells mediate several responses to hormone stimulation. To investigate the mechanism of the recognition of the uterus by the eosinophils, the present study describes the effect of a blockade of the rat reticulo-endothelial system with colloidal carbon on oestrogen-induced uterine eosinophilia, and other responses to oestrogen stimulation that, it has been suggested, are mediated by eosinophils.

In the absence of oestrogen colloidal carbon induced an increase in the number of eosinophils in mesometrium but not in endometrium with myometrium, and a slight oedematous reaction in deep endometrium. Colloidal carbon abolished the oestrogen-induced increase in the number of eosinophils in endometrium with myometrium and drastically decreased the oestrogen-induced increase in uterine wet weight and the endometrial oedematous responses 6 h after the administration of oestrogen.

The present results agree with the hypothesis that most uterine water imbibition is mediated by eosinophils and suggest a possible mechanism for the interaction of colloidal carbon with eosinophil migration to the uterus.

J. Endocr. (1986) 109, 89–95

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A. Armario, C. Restrepo, J. Hidalgo and A. Lopez-Calderón

ABSTRACT

Pituitary-adrenal, pituitary-gonadal and prolactin responses to acute stress (restraint) were studied in peripuberal and adult male rats. The pituitary-adrenal response to restraint stress did not differ in peripuberal and adult rats. Prolactin increase during stress was less marked in peripuberal animals. While an increase in LH during stress was observed in adult rats, peripuberal animals did not respond to stress. Testosterone levels were also lower in peripuberal than in adult rats. Diminished LH and prolactin responses to stress in peripuberal rats did not appear to be due either to increased pituitary-adrenal activity or to altered pituitary responsiveness to LHRH and dopamine respectively. Peripuberal rats were also more sensitive to the action of morphine on LH and prolactin release than were adult rats, suggesting that endogenous opioids may be involved in the LH and prolactin responses to acute stress. Differences in the maturation of central mechanisms rather than in pituitary response appear to be responsible for the differing responses to acute stress.

J. Endocr. (1987) 112, 9–13

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M Balbín, A Fueyo, J M López, I Díez-Itza, G Velasco and C López-Otín

Abstract

We have examined the expression of the murine counterpart of human collagenase-3, a matrix metalloproteinase produced by breast carcinomas, in the course of processes which involve extensive tissue remodeling. By using Northern blot analysis, we have found that collagenase-3 is expressed in the rat ovary, but not in the remaining analyzed tissues including brain, kidney, liver, lung, mammary gland, uterus, bladder, heart, intestine, prostate, spleen, testis and thymus. Collagenase-3 mRNA was detected at high levels in rat ovaries at proestrus and estrus, was at a minimum at metestrus and started to increase during diestrus through to proestrus. In addition, collagenase-3 was also detected on day 21 of pregnancy, which is approximately one day before parturition. However, no significative expression was detected in RNA from ovaries taken immediately after parturition, or on days 1, 5 or 30 postpartum. Northern blot analysis also revealed that collagenase-3 was not expressed at significant levels, compared with ovarian expression, in the uterus or in the mammary gland during pregnancy or after parturition. When follicular granulosa cells were separated from residual ovarian tissue and their RNA was analyzed by Northern blot, it was seen that collagenase-3 was not expressed by the granulosa cells but was present in the residual tissue containing interstitial and thecal tissues, growing follicles and corpora lutea. Immunohistochemical studies also confirmed, at the protein level, the localization of collagenase-3 in rat ovary. Gonadotropic stimulation of ovulation in immature rats by priming with pregnant mare's serum gonadotropin and stimulation with human chorionic gonadotropin failed to induce the expression of collagenase-3, suggesting that additional factors which are not present in the immature stimulated rats are needed for completely effective induction of the expression of this matrix metalloproteinase. On the basis of these results, together with the comparative analysis of expression of different matrix metalloproteinases in the rat ovary, we propose that collagenase-3 is a major ovarian metalloproteinase potentially involved in ovarian function during the reproductive cycle.

Journal of Endocrinology (1996) 149, 405–415

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A Martinez, R Pio, J Lopez and F Cuttitta

Adrenomedullin (AM) is a ubiquitous peptide hormone which, among other functional roles, reduces insulin secretion in the pancreas. Recently we have described the interaction between AM and the complement regulator protein factor H, which results in mutual modulation of their respective functions. Here we identify the expression of factor H in the beta cells of the rat pancreatic islets by immunohistochemistry and multiple immunofluorescence followed by confocal microscopy. In addition, double immunogold staining under the electron microscope showed coexistence of insulin and factor H immunoreactivities within the same secretory granules; interestingly, factor H staining was found in the electron-lucent haloes whereas the insulin antibody labeled preferentially the dense cores. The existence of factor H mRNA in the pancreas was confirmed by RT-PCR and in situ hybridization. The function of factor H in the pancreas was investigated with an insulin secretion assay. Addition of factor H to freshly isolated islets in the presence of AM resulted in a further reduction in insulin secretion with a concomitant elevation of cAMP, suggesting that factor H increases AM function in glucose homeostasis. The expression of factor H in the pancreas may play other important roles such as protection against complement-mediated cell lysis.

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SA Price, I Pochun, S Phaneuf and A Lopez Bernal

The precise factors involved in the transition of the relaxed pregnant uterus to the contractile state at the onset of parturition remain unclear, but it is accepted that cAMP-generating pathways contribute to uterine relaxation. We have previously reported an increased expression of the adenylyl cyclase (AC)-stimulating protein Galphas in human myometrium during gestation, with a corresponding increase in GTP-stimulated AC activity. However, little is known about the predominating AC isoforms expressed during pregnancy. This information is important, because although all AC isoforms are stimulated by Galphas, their regulation by other signalling molecules is very different. In the present study we have identified the isoforms of AC expressed in both pregnant and non-pregnant myometrium by mRNA analysis and immunoblotting. mRNA encoding for AC I, II, III, VIII and IX was present in non-pregnant and pregnant myometrium, and in cultured myometrial cells. Differing levels of AC protein could be detected in myometrial plasma membranes, with decreased levels of Group 1 (isoforms I, III and VIII) and Group 4 (IX) ACs allied with increased levels of Group 2 (II, IV and VII) and 3 (V and VI) ACs during pregnancy. These findings imply a role for Group 2-activating pathways, e.g. G-protein betagamma-subunits and protein kinase C, in the maintenance of uterine quiescence, whilst suggesting a lesser involvement of calcium-calmodulin complex, an activator of Group 1 AC isoforms, in uterine relaxation during gestation. These data may provide an alternative pharmacological approach for the attenuation of preterm labour.

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CB Brenninkmeijer, SA Price, A Lopez Bernal and S Phaneuf

There is evidence for hormonal receptor desensitisation in human myometrium, but little is known about the mechanisms involved in the loss of myometrial response to agonists such as beta(2)-adrenergic agonists, prostaglandin gamma and oxytocin. It is well known that the receptors for these hormones are coupled to G-proteins. The first step of receptor desensitisation is the phosphorylation of activated receptors by a G-protein-coupled receptor kinase (GRK). GRKs are members of a multigene family and the various subtypes differ in their localisation, regulation and mode of action. We have used Western blotting and reverse transcription PCR to identify the GRKs present in human myometrium from pregnant and non-pregnant women as well as in cultured human myometrial cells. We have found that human myometrium expresses the GRK subtypes 2, 4gamma, 5 and 6. On the other hand, GRK3 and the isoforms GRK4alpha, beta and delta were not found in myometrial tissue. Our data indicate that GRK2 is only expressed in pregnant term myometrium and is not found in non-pregnant tissue. Moreover, GRK6 appears to be expressed at a much higher level in pregnant term tissue than in non-pregnant myometrium. Our observations suggest that GRK2 and GRK6 may contribute to the regulation of uterine contractility at term. Further work is necessary to determine whether GRKs and receptor desensitisation play a role in disorders of uterine contractility.