Biomedical subjects

A Giaid

Publications and source records attributed to A Giaid.

84 records · Page 5Linked to original sources

PubMed1990

Effect of endocrine manipulation on anterior pituitary galanin in the rat.

Galanin is widely distributed throughout the rat neural and endocrine system. The highest concentrations are found in the anterior pituitary, and it can influence classical pituitary hormone secretion. The effects of endocrine manipulation on pituitary galanin content, mRNA, and immunostaining have been investigated in the rat. In females, medical (39 +/- 4 fmol/gland), surgical (33 +/- 2), or combined (28 +/- 6) castration resulted in a highly significant decrease in galanin content (control, 223 +/- 14; P less than 0.0001). Estrogen in physiological and pharmacological doses produced a significant increase in galanin content (368 +/- 14 and 373 +/- 13, respectively; P less than 0.01) associated with an increase in galanin mRNA content. In the male, high dose dexamethasone and thyroidectomy caused a fall in galanin content, while galanin mRNA levels showed a rise and fall, respectively. Adrenalectomy caused a rise in galanin content, while adrenalectomy and castration produced a dramatic decrease in tissue galanin content. No change in galanin mRNA was observed in these groups. Galanin immunostaining paralleled the results of tissue content in all groups examined, except in the medically castrated group, in which there was some intragroup variation in staining patterns. In normal and high-dose estrogen-treated females, galanin expression was seen mainly in lactotrophs, with a small number of somatotrophs and thyrotrophs staining. In the male, galanin expression was confined to somatotrophs and thyrotrophs. Galanin mRNA was localized at the cellular level by in situ hybridization. In the normal pituitary only scattered lactotrophs contained message, while in high-dose estrogen-treated animals the number of positive cells, mostly lactotrophs, was vastly increased. Thus, the cellular localization of galanin immunostaining varies between the sexes. Galanin peptide and mRNA levels in the pituitary are powerfully influenced by endocrine status.

Adrenalectomy↗

PubMed1990

Localization of immunoreactivity for calcitonin gene-related peptide in the rat anterior pituitary during ontogeny and gonadal steroid manipulations and detection of its messenger ribonucleic acid.

Localization of calcitonin gene-related peptide (CGRP) expression in the rat anterior pituitary and its changes during ontogeny and after gonadal steroid manipulations were studied by immunocytochemistry, RIA, and in situ hybridization. Colocalization studies and the combined use of immunocytochemistry and in situ hybridization revealed that CGRP immunoreactivity is localized mainly in gonadotropes and alpha- and beta-CGRP messenger RNAs were detected in CGRP-immunoreactive cells. Immunoreactivity for CGRP also was detected in nerve fibers and colocalized with substance P immunoreactivity. Cells immunoreactive to CGRP antiserum were first detected in fetal rats at gestational day 18, and the incidence considerably increased between postnatal days 5 and 14. CGRP immunoreactivity was low in control adults of both sexes and in pregnant and ovariectomized females but increased in lactating, estrogen-supplemented ovariectomized and high-dose estrogen-treated females, and in high-dose estrogen-treated and castrated males. Testosterone supplement suppressed the effect of castration on CGRP immunoreactivity in males. Quantities of extractable immunoreactive CGRP under conditions of estrogen manipulation corresponded well to the immunocytochemical findings (females: controls, 96.4 +/- 13.1 fmol/gland; ovariectomized, 107.6 +/- 19.2; high-dose estrogen-treated, 212 +/- 23.0; estrogen-supplemented ovariectomized, 680 +/- 42.1). The present study suggests that pituitary CGRP is synthesized and stored in gonadotropes, is modulated by gonadal steroids, and may have a functional link with gonadotropins.

Aging↗

PubMed1989

Detection of human probombesin mRNA in neuroendocrine (small cell) carcinoma of the lung. In situ hybridization with cRNA probe.

The production of human bombesin (gastrin-releasing peptide), a peptide with mitogenic action, is a recognized feature of neuroendocrine (small cell) carcinoma of the lung. However, immunostaining of bombesin is not always possible in these tumors, probably because of poor storage mechanisms or rapid release of hormone. Molecular biological analysis of the gene encoding human bombesin has revealed the DNA sequence of human pro-bombesin. We have used in situ hybridization to study the expression of the human bombesin gene at the cellular level in small cell carcinoma of the lung. Probombesin cDNA was subcloned in pSP64 vector, linearized with Bam HI and transcribed in the presence of phosphorus 32(32P)-cytosine triphosphate (CTP) and SP6 polymerase. The cRNA probe was applied to tissue sections (from six cases of small cell carcinoma of the lung, freshly fixed in 4% paraformaldehyde), cell culture preparations (two different cell lines of small cell carcinoma), and cytologic specimens (smears of cells from three different cases of small cell carcinoma). Hybridization of probombesin mRNA was detected in tumor cells in all samples. Specificity of the signal was determined by control experiments, including the use of a probe which has a sequence identical to probombesin mRNA. Our results provide evidence for the expression of the bombesin gene in small cell carcinoma of the lung at a cellular level and show that probombesin mRNA is highly expressed in these tumors.

Bombesin↗

PubMed1989

Non-isotopic RNA probes. Comparison between different labels and detection systems.

Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive 35S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.

Allylamine↗

PubMed1989

Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia.

The localization of endothelin 1 mRNA and endothelin-like immunoreactivity was investigated in samples of neurologically normal nervous system tissue from 10 adults by using in situ hybridization and immunocytochemistry. Tissue sections of spinal cord and dorsal root ganglia were hybridized with an 35S-radiolabeled endothelin 1 complementary RNA probe. Autoradiograms showed labeled neurons in the spinal cord (laminae IV-VI and many motoneurons) and numerous small and large neurons in the dorsal root ganglia. Endothelin 1 transcripts were also found in association with the endothelial layer of some blood vessels in the white matter of the spinal cord. A similar distribution of immunoreactivity was seen using three antisera to endothelin 1, but fewer cells were immunostained than were labeled after the hybridization. Two other endothelin 1 antisera immunostained the endothelial lining of blood vessels in the spinal cord white matter but not neurons. In the ganglia, endothelin 1 transcripts were localized to all cells expressing beta-preprotachykinin and most expressing calcitonin gene-related peptide mRNAs; in the majority of the motoneurons, coexistence of endothelin 1 and calcitonin gene-related peptide mRNAs was noted. There was a similar pattern of coexistence for respective peptide immunoreactivities in the ganglia and spinal cord. The expression of endothelin 1 mRNA in distinct neuronal cell types suggests that this peptide plays a part in neural transmission/modulation and adds a further dimension to the known vascular-related actions of endothelin 1.

Endothelins↗

PubMed1988

Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat.

Calcitonin gene-related peptide (CGRP) mRNA was localised to neurones of the dorsal root ganglia and motoneurones of the ventral horn in man and rat. Presence of alpha- and beta-CGRP mRNA was confirmed by Northern blot analysis of rat tissues which showed alpha-CGRP was the predominant gene. The distribution of CGRP gene transcripts corresponded with neurones displaying CGRP immunoreactivity in the ganglia of both species and in the rat ventral horn. In man few motoneurones were immunoreactive despite many expressing CGRP mRNA. In situ hybridisation revealed not only sensory but also motor neurones are sites of CGRP manufacture. Thus in conjunction with other evidence the present study reinforces the proposed muscle trophic role for this peptide.

Aged↗

PubMed1988

Localisation of mRNA and co-expression and molecular forms of GRP gene products in endocrine cells of fetal human lung.

The presence of bombesin (gastrin-releasing peptide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP- and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using region-specific antisera to pro-GRP. Additional antisera to chromogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining using antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro-GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreactive for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with immunocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-localised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Gel↗

PubMedDate not supplied

Increased reactivity to endothelin of pulmonary arteries in long-term post-obstructive pulmonary vasculopathy in rats.

Post-obstructive pulmonary vasculopathy (POPV) describes the constellation of findings following chronic ligation of one pulmonary artery, including bronchial angiogenesis, pulmonary vasculopathy and increased vasoreactivity to pharmacologic agents. Previously, we found in rats with POPV of 28 days duration that maximal responses to endothelin-1 (ET-1) and ET-3 of pulmonary arteries but not veins were increased, and that relaxation of arteries to ET-1 was reduced, attributable to an elevated proportion of ETA over ETB receptors. To determine the reactivity of pulmonary vessels and airways to ET in protracted POPV, we ligated the left main pulmonary artery of nine rats. Eleven months later, using a lung explant technique, we compared contractile responses of pulmonary vessels and airways to ET-1 and ET-3 in POPV lungs with controls. Morphometric measurements were made on the vessels, and immunoreactivity to ET-1 and endothelin converting enzyme (ECE-1) studied. We found contractile responses to ET-1 and ET-3 significantly increased in arteries, but not veins or airways. Morphometry showed arteries and veins had reduced diameters with muscle thickening only in veins. Expression of ET-1 and ET-3 in vascular endothelial cells and airway epithelial cells were not altered significantly. Our data suggest that protracted POPV selectively enhanced the contractility of pulmonary arteries to ET, and is not attributable to medial muscle thickening.

Animals↗

PubMedDate not supplied

Newborn piglet lungs release endothelin-1: effect of alpha-thrombin and hypoxia.

Endothelin-1 (ET-1) is a 21 amino acid vasoconstrictor peptide produced by endothelial cells, the expression of which is modulated by a variety of vasoconstrictors, vasodilators, and inflammatory mediators. Hypoxia has been shown to increase ET-1 expression and release in cultured endothelial cells from the systemic circulation, but reports are contradictory regarding the pulmonary circulation. In this study, the release of ET-1 and its cellular localization in the isolated perfused newborn piglet lung were examined under control conditions and after stimulation with hypoxia or alpha-thrombin (positive control). In the control condition, perfusion pressure remained stable during the study period, and a progressive increase in levels of immunoreactive ET-1 (irET-1) was noted. When alpha-thrombin was added to the perfusion fluid, a slow gradual increase in perfusion pressure was produced and the levels of irET-1 were significantly greater than those measured in the control preparations. Finally, hypoxia produced a significant increase in the perfusion pressure; however, the release of irET-1 did not differ significantly from the control, if anything, the net release across the lung was diminished. In all conditions, immunocytochemistry using antiserum to human-porcine ET-1 revealed the presence of high ET-1-like immunoreactivity in epithelial cells of bronchi, bronchioles, and terminal bronchioles. In addition, endothelial cells of large and medium-size pulmonary arteries were only moderately immunoreactive for ET-1. These findings indicate that the neonatal pig lung can produce and release ET-1, and that its release can be increased by certain stimuli like alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMedDate not supplied

Endothelin-1-like immunoreactivity in postobstructive pulmonary vasculopathy.

Postobstructive pulmonary vasculopathy (POPV) is produced by chronic ligation of one pulmonary artery and results in bronchial collateral vessel proliferation and pulmonary arterial abnormalities. The role of endothelin-1 (ET-1), a potent vasoconstrictor peptide, was investigated in a canine model of POPV using radioimmunoassay and immunohistochemistry. The left main pulmonary artery of 9 dogs was ligated, and 3 months (n = 3) and 15 months (n = 6) later, ET-1 levels were measured by radioimmunoassay of plasma samples from left and right pulmonary arteries and veins. In addition, tissues from control and ligated lungs were fixed in Bouin's solution embedded in paraffin and stained with antiserum to ET-1. Plasma ET-1 levels distal to the ligation were not different from those of the control pulmonary artery (1.7 pg/ml in the ligated lungs vs. 1.4 pg/ml in the controls; NS). ET-1-like immunoreactivity was localized mainly to the epithelium in both control and ligated lungs, but the pulmonary arteries and the new bronchial vessels stained more intensely in the ligated lungs. The sections immunostained with ET-1 antiserum preabsorbed with synthetic ET-1 did not stain. These results suggest that ET-1 may play a role in the bronchial neovascularization and the pulmonary arterial thickening characteristic of POPV.

Animals↗

PubMedDate not supplied

Cyclooxygenase-2 and nuclear factor-kappaB in myocardium of end stage human heart failure.

Over the past three decades, numerous studies have shown diverse functional and morphological effects for prostanoids on cardiac myocytes with levels of prostanoids elevated in congestive heart failure. We examined the expression of cyclooxygenase-2 (COX-2) and activation of nuclear factor-kappaB (NF-kappaB) in failing human hearts. Expression of COX-2 and activation of NF-kappaB were examined in myocardial tissues from 27 patients with end stage heart failure, 2 septic patients, and 8 normal control subjects. There was little or no expression of COX-2 and NF-kappaB in the control hearts. There was abundant expression of COX-2 mRNA and protein in myocytes and inflammatory cells in areas of fibrotic scar compared with regions of normal morphology in all cases of heart failure, except in the cases with sepsis which showed abundant COX-2 throughout the myocardium. Sites of NF-kappaB activation were associated with those of COX-2 expression. We concluded that COX-2 expression and NF-kappaB activation are elevated in patients with congestive heart failure. Cellular induction of both molecules is associated with sites of inflammation and scar formation. (c)1999 by CHF, Inc.

Journal Article↗

PubMedDate not supplied

Improved outcome of rat lung transplantation with modification of the nonsuture external cuff technique.

BACKGROUND: The development of lung transplantation in inbred rats has simplified immunologic studies concerning lung allografts. Unfortunately, up to now, such models have been limited by the high level of surgical expertise required. METHODS: In this study 92 rats underwent left lung transplantation with a simplified cuff technique. The feasibility and reliability of this technique was determined by examining graft patency through the use of gross and histologic examination, angiography, and bronchography. RESULTS: On gross examination, grafts looked well perfused and ventilated. Histologic examination showed little evidence of foreign body reaction against the Teflon cuff devices, and angiography showed no abnormalities; however, bronchography did show signs of airway stricture at the site of the anastomosis. CONCLUSION: We conclude that the nonsuture external cuff technique with Teflon devices represents a reliable, safe, and fast method for performing orthotopic left lung transplantation in the rat.

Anastomosis, Surgical↗