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Adrenomedullin Expression and Function in the Rat Carotid Body

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Adrenomedullin Expression and Function in the Rat Carotid Body 95 Adrenomedullin expression and function in the rat carotid body A Martínez, L Saldise1, M J Ramírez2, S Belzunegui1, E Zudaire, M R Luquin1 and F Cuttitta Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 13N262, Bethesda, Maryland 20892, USA 1Department of Experimental Neurology, University of Navarra, 31080 Pamplona, Spain 2Department of Pharmacology, University of Navarra, 31080 Pamplona, Spain (Requests for offprints should be addressed to A Martínez; Email: [email protected]) Abstract Adrenomedullin (AM) immunoreactivity has been found 60 nM AM/g body weight into the tail vein of the rats did in granules of the glomus (type I) cells of the carotid bodies not induce statistical differences in DA release from the in rats. The identity of these cells was ascertained by carotid bodies. Exposure of the oxygen-sensitive cell line colocalization of immunoreactivities for AM and tyrosine PC-12 to hypoxia elicited an increase in AM mRNA hydroxylase in their cytoplasm. Exposure of freshly iso- expression and peptide secretion into serum-free condi- lated carotid bodies to synthetic AM resulted in a tioned medium. Previous data have shown that elevation concentration- and time-dependent degranulation of of AM expression under hypoxia is mediated through glomus cells as measured by dopamine (DA) release. DA hypoxia-inducible factor-1, and that exposure of chroma- release reached a zenith 30 min after exposure to AM ffin cells to AM results in degranulation. All these data (94·2% over untreated controls). At this time-point, the suggest that AM is an important autocrine regulator of response to AM was similar to the one elicited by 5 min of carotid body function. + exposure to 100 mM K . Nevertheless, injection of 1 µl Journal of Endocrinology (2003) 176, 95–102 Introduction pathological conditions such as heart failure, myocardial infarction, hypertension, and septic shock (Eto et al. 1999). Adrenomedullin (AM) is a 52 amino acid peptide contain- Conversely, intracerebroventricular administration of AM ing an internal disulfide bond and a C-terminal amide induces hypertension and tachycardia in conscious rats group. It was originally isolated from a pheochromocytoma (Saita et al. 1998). In addition, when microinjected into and shown to elevate cAMP in platelets (Kitamura et al. the area postrema, AM increases blood pressure and heart 1993). Many functions have been ascribed to AM. This rate (Mark et al. 1997). These observations thus indicate peptide can act as a vasodilator, bronchodilator, growth that AM influences blood pressure in a differential manner factor, regulator of hormone secretion, neurotransmitter, depending on the site of action. antimicrobial agent, and a controller of renal function, Carotid bodies are a small pair of highly vascularized and among others (Hinson et al. 2000, López & Martínez well-perfused organs located at each carotid artery bifur- 2002). cation. They are sensory organs that detect decreases in The most characteristic activity of AM is the induction arterial blood oxygen and react by stimulating the nucleus of an intensive, long-lasting dose-dependent hypotension tractus solitarius in the brain stem. This nucleus can in humans, rats, rabbits, dogs, cats, and sheep. AM dilates precipitate a wide array of systemic reflex responses, resistance and capacitance vessels in the kidneys, heart, including ventilatory and cardiovascular acclimatization. A brain, lung, hindlimbs, and mesentery. Moreover, AM bulk of evidence suggests that glomus (type I) cells are the elicits relaxation of ring preparations of the aorta and initial site of transduction and that they release transmitters cerebral arteries. An intravenous injection of human AM in response to hypoxia, which in turn cause depolarization to conscious sheep causes a dose-dependent fall of blood of nearby afferent nerve endings. Acute hypoxia causes a pressure, an increase in heart rate and cardiac output with rapid response within milliseconds, which is mediated by a small reduction in stroke volume, as well as a marked oxygen-sensitive K+ channels located in the glomus cell decrease in total peripheral resistance (Parkes 1995). In membrane. On the other hand, responses to chronic human coronary arteries, AM elicits vasodilatation through hypoxia are much slower and include changes in gene specific AM receptors and by enhancing production of expression profiles that are responsible for the phenotypic nitric oxide (Terata et al. 2000). AM levels increase under changes observed in pathophysiological situations such as Journal of Endocrinology (2003) 176, 95–102 Online version via http://www.endocrinology.org 0022–0795/03/0176–095 2003 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/03/2021 12:35:09AM via free access 96 A MARTIuNEZ and others · Adrenomedullin in the carotid body sleep apneas, apneas in premature infants, asthma, or concentration of 1:1000. The next day, the sections were pulmonary fibrosis. Some of these hypoxia-induced incubated for 1 h with a mixture of Bodipy-goat anti- changes in gene expression include elevation in the levels rabbit (Molecular Probes, Eugene, OR, USA), and Texas of dopamine (DA), norepinephrine, nitric oxide, and red-donkey anti-mouse (Jackson Immunoresearch Labs), endothelin, and a decrease in the levels of substance P both at a final concentration of 1:200. Counterstaining (López-Barneo et al. 2001, Prabhakar et al. 2001). with DAPI (Molecular Probes) was performed for 10 min. AM is expressed by chromaffin cells of the adrenal After thorough washes, the slides were mounted in medulla (Kapas et al. 1998), which share a common GelMount (Biomeda, Foster City, CA, USA) and embryonic origin with glomus cells and have similar observed with a Zeiss laser scanning microscope 510, mechanisms of oxygen sensing and response (Millhorn equipped with four lasers. et al. 1996) and are often used as physiological models for carotid body studies. AM expression is upregulated under hypoxic conditions through hypoxia-inducible factor-1 Analysis of DA secretion (HIF-1) (Garayoa et al. 2000) and has been found in many Freshly isolated carotid bodies (12–16 per treatment) were structures of the central nervous system (Serrano et al. incubated with synthetic AM (Peninsula Labs, San Carlos, 2000). In addition, exposure of the rat brain to ischemia CA, USA) in a time-course (5–60 min) and a dose– reperfusion results in a dramatic increase in AM expression response (108 to 105 M) manner. Individual carotid in both neural and vascular structures of the brain (Serrano bodies were immersed in 100 µl 0·4 M perchloric acid, et al. 2001). Based on these facts we decided to study 1 mM EDTA, and 0·1% sodium metabisulfite and whether AM is expressed in the carotid body and, if so, vortexed for 1 min to extract DA as described (Verbiese- whether it influences carotid body physiology. Genard et al. 1983). DA contents in the extraction samples (20 µl) were determined using high-performance liquid chromatography with electrochemical detection as pre- Materials and Methods viously reported (Kilts et al. 1981). The mobile phase composition was 0·1 mM Na2HPO4, 0·1 M citric acid, Animals and surgical procedure 0·74 mM octansulphonic acid, 1 mM EDTA, and 14% methanol, pH 3·4. DA contents were calculated by Experiments were performed using carotid bodies from extrapolation against a standard curve (125–500 pg/20 µl adult male Wistar rats (body weight (BW) 240–280 g) DA). A group of 12 carotid bodies was incubated with which were housed under standard conditions of 50% 100 mM KCl for 5 min, to check the validity of our humidity, 16 liters/min air exchange, and a 12 h experimental procedure by studying the response of the light:12 h darkness cycle, with food and water provided preparation to a well-known depolarizing stimulus ad libitum. Animals were anesthetized by intraperitoneal (Rosenbland & Nilsson 1993). Different sets of samples injection of 80 mg/kg ketamine (Ketolar; Park-Davis, were used for each incubation time. Madrid, Spain) and 10 mg/kg xylaxine (Rompum; Bayer, For the in vivo study, 18 animals received 1 µl 60 nM Madrid, Spain). Carotid bodies were removed from the AM/g BW intravenously. After 30, 60, and 90 min of the carotid bifurcation, cleaned of surrounding connective injection, six rats per time-point were anesthetized as tissue and either fixed for immunodetection (see below) or before and the carotid bodies dissected out. DA content treated as indicated and kept frozen at 80 C until analysis was performed as for the in vitro experiments. In extracted. All procedures were approved by the animal one control group (n=6), the carotid bodies were dissected ethics committee of the University of Navarra. immediately after being injected with saline. To take into account variations due to stress after injections, a second group of controls (n=6) were obtained from animals killed Double immunofluorescence and confocal microscopy 90 min after the saline injection. Carotid bodies from four animals were fixed in 4% paraformaldehyde for 30 min, embedded in OCT com- Cell culture and Northern blot analysis pound (Tissue-Tek; Miles Inc., Elkhart, IN, USA) and frozen in liquid nitrogen. Sections (20 µm thick) were Cell line PC-12 has been previously characterized as an blocked with a mixture of 3% donkey and 3% goat normal oxygen-sensing cell line and has been frequently used as a sera (Jackson Immunoresearch Labs, West Grove, PA, model for carotid body function (Millhorn et al. 1996). USA) and then exposed to a mixture of two primary This rat cell line was obtained from ATCC (Manassas, VA, antibodies obtained from different species, overnight at USA) and grown in RPMI 1640 medium supplemented 4 C. The mixture consisted of a previously characterized with 10% horse serum and 5% fetal bovine serum (Life rabbit antibody against AM (Martínez et al.
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