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Carotid Body Remodelling in l -NAME-Induced Hypertension in the Rat
J. Comp. Path. 2012, Vol. 146, 348e356
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EXPERIMENTALLY INDUCED DISEASE
Carotid Body Remodelling in L-NAME-Induced Hypertension in the Rat A. S. Felix, V. N. Rocha, A. L. R. Nascimento and J. J. de Carvalho Laboratory of Ultrastructure and Tissue Biology, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Summary The carotid body (CB) is a chemoreceptor organ located at the bifurcation of the common carotid artery. It is made up of the carotid glomus, a structure containing type 1 cells surrounded by type 2 cells. The aim of this study was to evaluate the morphological changes of the CB and carotid glomus in the rat model of L-NAME-induced hypertension. Male Wistar rats were divided in two groups: control untreated rats (C) and rats receiving L-NAME 40 mg/kg/day (LN) for 6 weeks. At the end of the experiment, the systolic blood pressure was 63% higher in the LN group compared with the C group. Morphometric analysis showed that the area of the CB was 29% greater in the LN group compared with the C group. The density of nuclei in the CB was similar between groups, but it was 31% less in the carotid glomus of the LN group. Cells in the CB of the LN group displayed cytoplasmic vacuolation and expressed several biogenic amines. There were more elastic fibres, proteoglycans and collagen fibres in the LN group compared with the C group. Immunohistochemistry showed increased expression of nuclear factor kB, substance P, vascular endothelial growth factor and neuronal nitric oxide synthase in the LN group, while expression of the protein gene product 9.5 was decreased. L-NAME alters cell morphology and the expression of extracellular matrix molecules in the CB and carotid glomus in rats with L-NAME-induced hypertension. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: carotid body; carotid glomus; hypertension; L-NAME
Introduction Systemic hypertension augments the risk of morbidity and mortality due to cardiovascular disease (Mayet and Hughes, 2003; Gkaliagkousi et al., 2010; Park et al., 2010; Wang et al., 2010). The endothelium plays a central role in regulating the vascular tone through the synthesis and release of vasoactive substances such as nitric oxide (NO), prostaglandin (PG) I2 and endothelium-derived hyperpolarizing factor (EDHF), as well as vasoconstrictor factors (Qin et al., 2000; Gkaliagkousi et al., 2010). Endothelial dysfunction in hypertension triggers an imbalance between the production and release of these factors, increasing the generation of reactive oxygen species and diminishing NO synthesis and bioavailability. L-arginine is the precursor of NO Correspondence to: J. J. de Carvalho (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2011.07.007
synthesis by NO synthase (NOS), an enzyme that exists in three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS) (Moncada and Higgs, 2006). Administration of L-arginine analogues such as L-NAME (hydrochloride NG-nitro-methylester-L-arginine) inhibits NOS activity and hence NO biosynthesis, leading to hypertension (Nakanishi et al., 1995; Swislocki et al., 1995; Fink et al., 1998; Bernatova et al., 1999; Fernandes-Santos et al., 2006). The carotid body (CB) is a chemoreceptor organ located at the bifurcation of the common carotid artery. The CB senses blood gas levels and regulates ventilation (Biscoe, 1971; Feng et al., 2008; Abudara and Eyzaguirre, 2008; Izal-Azcarate et al., 2008; Torres, 2009). It has a neural origin that can be characterized by the presence of the protein gene product 9.5 (PGP 9.5), a hydrolase found in neural and neuroendocrine tissues (Wilkinson, 2000). In rats, the CB consists of cellular aggregates (the carotid Ó 2011 Elsevier Ltd. All rights reserved.
Carotid Body Morphology in Hypertension
glomus) interspersed between blood vessels and nerve bundles. The carotid glomus has four to seven type 1 cells (or glomus cells) incompletely surrounded by two to three type 2 cells (or sustentacular cells) (Izal-Azcarate et al., 2008). Type 1 cells are characterized by the presence of numerous cytoplasmic vesicles containing dopamine, a biogenic amine, and gap junctions between adjacent cells (Morgan et al., 1975; Eyzaguirre, 2005). Afferent nerve endings establish synaptic contact with one or more type 1 cells (Rigual et al., 1991; Conde et al., 2006). Taquinin is an excitatory neurotransmitter that has a role in the brainstem-mediated response to CB stimulation. The tachykinin, P substance (PS) and the amino acid, L-glutamate, appear to be important excitatory neurotransmitters. PS acts on NK1 (PS receptor), a typical G protein-coupled receptor located in the afferent nerve endings of the CB (Mazzone et al., 1997). The extracellular matrix of the CB is composed of ground substance (i.e. glycosaminoglycans, proteoglycans and adhesive glycoproteins) and fibres (Evangelisti et al., 1984; Boudreau and Rabinovitch, 1991). However, data are scant regarding CB morphology in hypertension. It is known that there is a significant increase in CB volume in spontaneously hypertensive rats (SHRs) compared with normotensive Wistar Kyoto rats (Habeck et al., 1987). Similarly, New Zealand genetically hypertensive rats have fewer type 1 cells than control rats (Bee et al., 1989). Finally, abnormalities in dopamine production have been described in severe hypertension in man (Murphy et al., 2001). We hypothesize that systemic hypertension changes the normal morphology and protein expression of the CB. The aim of the present study was to characterize the morphological changes in the CB and carotid glomus in the rat model of L-NAME-induced hypertension.
Materials and Methods Animals and Sample collection
Three-month old Male Wistar rats were obtained from the State University of Rio de Janeiro (UERJ). Rats were kept under standard housing conditions (21 2 C, 60 10% humidity and a 12 h lightedark cycle) and received water and standard rodent chow (Nuvilab, Parana, Brazil) ad libitum. All procedures were approved by the Ethics and Research Committee of the Institute of Biology Roberto Alcantara Gomes/UERJ (Protocol 257/08) and were carried out in accordance with the conventional guidelines for the Care and Use of Laboratory Animals (US National Institutes of Health, revised 1996).
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Animals were divided into two groups (n ¼ 10): untreated rats (control group, C) or rats treated with L-NAME (SigmaeAldrich, St Louis, Missouri, USA) at a dosage of 40 mg/kg/day in their drinking water (L-NAME group, LN) (Bernatova et al., 1999; Kasal et al., 2008). Body mass and systolic blood pressure (SBP) were measured weekly throughout the 6 weeks of the experiment by plethysmographic flow (LETICA LE 5100; Panlab, Barcelona, Spain). At the end of the experiment, rats were anaesthetized with sodium pentobarbital (50 mg/kg by intraperitoneal injection). An incision was made in the neck to expose the carotid bifurcation and then the CB was rapidly freed from adherent adipose and connective tissue for isolation and fixation. Samples were collected from both sides of the neck and processed for light (n ¼ 6) and electron (n ¼ 4) microscopy. Microscopy
CB samples were fixed in 4% paraformaldehyde in 5 mM CaCl2 and in 0.1% glutaraldehyde and were processed routinely and embedded in paraplast plus (SigmaeAldrich). Sections (3 mm) were stained with haematoxylin and eosin (HE), Weigert’s resorcinfuchsin (Fullmer, 1960), picrosirius red (to demonstrate collagen fibres) (Puchtler et al., 1973) and alcian blue pH 2.5 (to demonstrate proteoglycans) (Whiteman, 1973). In order to study the vesicles containing biological amines, CB samples were examined with a laser scanning confocal microscope (LSM 510 Meta; Carl Zeiss, G€ottingen, Germany) using formaldehyde-induced fluorescence (FIF).
Immunohistochemistry
CB sections were incubated with primary antibodies specific for: vascular endothelial growth factor (VEGF; dilution 1 in 100; Santa Cruz Biotechnology, Santa Cruz, California, USA; sc-7269), nuclear factor kB (NF-kB; dilution 1 in 100; Santa Cruz Biotechnology; sc-114), substance P (PS; dilution 1 in 100; Chemicon, Billerica, Massachusetts, USA; AB1566), nNOS (dilution 1 in 50, Santa Cruz Biotechnology; sc-648) and PGP 9.5 (dilution 1 in 50; Abcam; AB8189). The reaction was amplified using a biotinestreptavidin complex system (LSAB Kit Universal; DakoCytomation, Glostrup, Denmark) and the immunolabelling was ‘visualized’ after incubation with 3, 30 diaminobenzidine tetrachloride (DAB; DakoCytomation). Sections were counterstained with Mayer’s haematoxylin.
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Electron Microscopy
CB samples fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and 0.25% tannic acid (Merck, Darmstadt, Germany) were post-fixed in osmium tetroxide 1% (SigmaeAldrich) and potassium ferrocyanide 0.8% in 0.1 M cacodylate buffer (pH 7.2) for 1 h and then dehydrated in acetone and embedded in Epon (Embed-812; Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Semithin sections (1 mm) were stained with toluidine blue and observed by light microscopy to select the areas of interest. Ultrathin sections (Leica Ultracut-UCT, Vienna, Austria) were contrasted with uranyl acetate and lead citrate and analyzed with a transmission electron microscope (Zeiss EM 906; Carl Zeiss, Oberkochen, Germany) at 80 kV. CB sections were fixed in paraformaldehyde 4%, processed and embedded in LR-White resin for immunoelectronmicroscopical analysis of expression of the enzyme tyrosine hydroxylase (TH). The secondary antibodies were conjugated to colloidal gold particles (10 nm). After immunolabelling, the sections were contrasted with uranyl acetate and lead citrate and analyzed by transmission electron microscopy (TEM) as above.
Fig. 1. Selection of the CB area chosen for measurement. Measurement was performed by the functions ‘clear outside’ and ‘measure’ of the Image J software.
the Student’s t-test. P # 0.05 was considered statistically significant (GraphPad Prism version 5.03, San Diego, California, USA).
Morphometry
Digital images were obtained using a digital camera (Olympus DP70, Tokyo, Japan) attached to a microscope (Olympus BX51). Sections stained with HE were used to quantify the density of nuclei in the CB and carotid glomus. Firstly, the boundary of the CB was outlined and its area was assessed (Image J software version 1.4, National Institutes of Health, Bethesda, Maryland, USA). Then, all nuclei (type 1 and type 2 cells) located inside the carotid glomus were quantified. The density of nuclei per CB (nuclei/mm2 of CB area) was calculated as: number of nuclei/image area (Fig. 1). Secondly, to quantify the density of nuclei in the carotid glomus, its boundary was first outlined and the area calculated. Then, the number of nuclei inside the carotid glomus was automatically calculated by the software. The density of nuclei per carotid glomus (nuclei/mm2 of carotid glomus area) was calculated as: number of cells inside the carotid glomus/carotid glomus area. Seven rats were analyzed per group and for each sample, 20 fields were examined, totalling 140 fields per group.
Results L-NAME increased the SBP and induced body weight loss in the LN group compared with the C group (Table 1). A difference in SBP was first noticed in the second week of the experiment and it increased in the following weeks. SBP reached 190 7.01 mmHg in the LN group compared with 122.2 2.51 mmHg in the C group (an increase of 63%). Body weight was similar between C and LN groups at the beginning
Table 1 Blood pressure, body mass and CB morphometry Control
L-NAME
P
117 5.8 190 7.01
NS 0.0001
265.4 13.0 306.0 14.9 152,400 12,030 5,877 361 4,936 378
NS 0.0002 0.0056 NS 0.0003
Statistics
Blood pressure (mm Hg) Initial 120 7.0 Final 122.2 2.51 Body mass (g) Initial 271.3 9.0 Final 375.6 24.2 106,000 7,181 CB area (mm2) 6,602 160 Nuclei/CB (mm2) 7,190 350 Nuclei/carotid glomus (mm2)
Data are shown as mean and standard error of the mean (SEM). Data were tested for normality. Differences between the C and LN groups were analyzed by
Data are shown as mean SEM, Student’s t-test. Initial refers to data assessed before the beginning of the experiment (week 0) and final are data on week 6. NS, non-significant.
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of the experiment, but it had decreased by 19% in LN group in the 6th week compared with the C group. The CB area increased in the LN group by 29% compared with the C group (Table 1). The density of nuclei in the CB was similar between the C and LN groups, suggesting CB cell hypertrophy rather than hyperplasia. Additionally, the density of nuclei in the carotid glomus was 31% less in the LN group compared with the C group. L-NAME-induced morphological changes in the carotid glomus are shown in Fig. 1. In the LN group, the regular morphology of the CB was lost as the carotid glomus cells were hypertrophic and had cytoplasmic vacuolation (Figs. 2a, b). The FIF technique allows the visualization of biogenic amines, and there was a greater amount of these in the LN group compared with the C group (Figs. 2c, d). Some electron-dense granules were
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found in the cytoplasm of cells of the carotid glomus in the C group, but these were more frequently seen in the LN group (Figs. 2e, f). The presence of vacuoles in the cytoplasm of the carotid glomus cells (Figs. 2e, f) was also evident by TEM. TEM analysis for expression of TH showed reduced expression of the enzyme in the cytoplasm of type 1 cells in the LN group compared with those cells in the C group. Colloidal gold particles were found in smaller numbers in the hypertensive group (LN) (Fig. 3). Morphological changes in the extracellular matrix are depicted in Fig. 4. The LN group displayed more elastic fibres (Figs. 4a, b) and proteoglycans (Figs. 4c, d) compared with the C group. The picrosirius red stain, without (Figs. 4e, f) or with (Figs. 4g, h) light polarization, revealed an increased number of collagen fibres in the LN group compared with the C group.
Fig. 2. CB structure and ultrastructure. (a, b) Photomicrographs. HE. Bar, 30 mm. (c, d) FIF; arrows indicate biogenic amines. Bar, 30 mm. (e, f) TEM of the CB. Bar, 2 mm. White arrows indicate the biogenic amines in the CB. Oval white arrows show the location of biogenic amines in cells of the carotid glomus.
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Fig. 3. Immunoelectronmicroscopy of the CB with primary anti-TH antibody and a secondary antibody conjugated to 10 nm colloidal gold particles. White arrows indicate the labelling in the cytoplasm of the CB type 1 cells. (a, b) Group C. (c, d) LN group.
The expression of five different proteins was analyzed by immunohistochemistry. L-NAME treatment increased the expression of NF-kB, substance P, VEGF and nNOS compared with the C group. PGP 9.5 was the only protein that had its expression diminished by L-NAME treatment. NF-kB was expressed in the extracellular matrix, while substance P and PGP 9.5 were found in the carotid glomus. VEGF was mainly expressed by glomus cells in the C group, but it was distributed in scattered fashion throughout the cytoplasm of glomus cells and blood vessels in the LN group. Finally, nNOS was more highly expressed in the carotid glomus and extracellular matrix in the LN group, a pattern not seen in the C group (Fig. 5).
Discussion The NOS inhibitor L-NAME has been used extensively as a means of inducing hypertension in animal models (Kasal et al., 2008). The results of the
present study have shown that L-NAME-induced hypertension alters the amount of extracellular matrix proteins and cellular morphology of the CB. The increase in elastic fibres, proteoglycans and collagen fibres correlates with the increased CB area in L-NAME-treated rats. Changes in protein expression are thought to reflect the increased blood pressure and sympathetic activity and probably occur in order to normalize the physiological function of the CB. CB volume increases in rats subjected to chronic hypoxia (Dhillon et al., 1984; Clarke et al., 2000) and the same phenomenon is observed in New Zealand genetically hypertensive rats (Bee et al., 1989). An enlargement of the CB was also noticed in the present study. This was due to a combination of enhancement of extracellular matrix deposition and hypertrophy of the carotid glomus cells. The extracellular matrix deposition was characterized as an increased amount of elastic fibres, proteoglycans and collagen. The density of nuclei in the carotid glomus was diminished, while the carotid
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Fig. 4. Photomicrographs showing the extracellular matrix of the CB. (a, b) Weigert’s resorcin-fuchsin stain. (c, d) Alcian blue stain. (e, f) Picrosirius red stain. (g, h) Picrosirius red stain under polarized light. Bars, 30 mm. Thin black arrows indicate the presence of elastic fibres around blood vessels. Oval black arrows indicate the presence of elastic fibres within the extracellular matrix of the CB. Diamond black arrows indicate the deposition of proteoglycans around blood vessels. White arrowheads indicate collagen fibres within the CB.
area was bigger, suggesting hypertrophy of the carotid glomus cells. NF-kB is a protein complex expressed in almost all animal cells and it controls DNA transcription. In the heart, NF-kB activation enhances cardiac remodelling and dysfunction following myocardial infarction (Timmers et al., 2009), angiotensin II-induced cardiac hypertrophy (Dai et al., 2010) and pressure overload-induced left ventricular hypertrophy (Hingtgen et al., 2010). We found increased expression of NF-kB in L-NAME-treated rats, thus this complex may be driving both extracellular matrix remodelling and the hypertrophy of carotid glomus cells. PGP 9.5 has been described in glomus type 1 cells (Kameda, 1996) and its expression in the CB was diminished by L-NAME treatment. This result is probably due to a reduction in the deubiquitination process and thus accumulation of unnecessary proteins for CB cellular metabolism. L-NAME-treated rats had greater amounts of biogenic amines within the CB. The observed electron-dense granules likely represent biogenic amines, as does the fluorescence depicted in Figs. 1c, d. Biogenic amines such as catecholamines are particu-
larly found in vesicles located in the cytoplasm of type 1 cells of the glomus (Shirahata et al., 2007). Studies of patients with obstructive sleep apnoea and cardiovascular morbidity, particularly systemic hypertension, show a significant increase in the production and circulation of catecholamines during apnoea (Fletcher et al., 1987; Smith, 2007). This neurotransmitter is released and increases sympathetic activity in order to maintain blood pressure during sleep disorders and hypoxia (Chiocchio et al., 1966; Fidone et al., 1982). Glomus type 1 cells also produce the excitatory neurotransmitter SP. During hypoxia there is an increased demand, suggesting that nerves containing the SP receptor are involved in the chemosensory response and vascular dilation (Hallberg and Pernow, 1975; Kusakabe et al., 1998, 2003). In the present study, rats subjected to NOS inhibition had increased expression of SP, suggesting that this molecule is being used as a chemosensory stimulus and thus might be an important vasodilator to improve blood circulation and keep oxygen reaching the tissues. Dopamine, a catecholamine, is produced from the non-essential amino acid tyrosine and is transformed into L-DOPA by TH. Type 1 cells have been
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Fig. 5. Immunohistochemical analysis of the CB. (a, b) NF-kB. (c, d) PS. (e, f) protein PGP 9.5. (g, h) VEGF. (i, j) nNOS. (k, l) Negative control. Bars, 30 mm.
identified, in previous studies, as the main storage site for catecholamines in the CB. Dopamine is probably a neurotransmitter of afferent fibre discharge (Fidone and Gonzalez, 1982). The release of this neurotransmitter might alter stimulatory capacity of type 1 cells (Wang et al., 1998). Experimental studies have shown that levels of dopamine and noradrenaline decrease after an episode of acute hypoxia, suggesting that these molecules are released by type 1 cells (Rigual et al., 1991) in order to normalize the levels of blood oxygenation. The results of the present study show a reduction in TH in the hypertensive group (LN) compared with the control group (C) in response to L-NAME, even though there was an increase in catecholamines in the CB of the LN group. An explanation for this finding would be that the hypertension induced by L-NAME increases the production and storage of these biogenic amines within
the cytoplasm of the type 1 cells before the remodelling of the organ, with loss of their structure and reduction in TH level. Available data indicate that NOS isoforms produce NO within diverse cell types in the CB, such as the vascular endothelial cells, parasympathetic neurons, afferent nerve terminals and glomus type 1 cells (Ye et al., 2002; Yamamoto et al., 2006). The neuronal isoform of NOS is contained in a dense plexus of afferent nerve fibres that terminate near oxygen-sensitive type 1 cells in the CB of rats. Additionally, a distinct group of nNOS-positive parasympathetic neurons have been shown to innervate the CB vasculature (Wang et al., 1994, 1995). In the present study, there was increased expression of nNOS in glomus cells and in components of the extracellular matrix of LNAME-treated rats. VEGF increases vascularization and restores oxygen supply when blood flow to
Carotid Body Morphology in Hypertension
tissues becomes inadequate (Ferrara et al., 1992; Prabhakar et al., 2009). VEGF expression in CB also increased following inhibition of NOS and a similar result was found by Tipoe and Fung (2003) in rats exposed to chronic hypoxia. Hence, the increased expression of nNOS and VEGF in L-NAME-treated rats indicates an attempt to increase the synthesis of the vasodilator NO and tissue perfusion in the CB. In conclusion, the present study has shown that induction of hypertension in rats treated with L-NAME changes cellular morphology and the expression of extracellular matrix proteins in the CB and carotid glomus.
Acknowledgments The authors are grateful to the Brazilian Council of Science and Technology (CNPq), the Rio de Janeiro State Foundation for Scientific Research (FAPERJ), the Council for the Improvement of Graduate Personnel (CAPES) and the State University of Rio de Janeiro (UERJ).
Conflict of Interests The authors disclose no conflict of interest.
References Abudara V, Eyzaguirre C (2008) Mechanical sensitivity of carotid body glomus cells. Respiratory Physiology and Neurobiology, 161, 210e213. Bee D, Barer G, Wach R, Pallot D, Emery C et al. (1989) Structure and function of the carotid body in New Zealand genetically hypertensive rats. Quarterly Journal of Experimental Physiology, 74, 691e701. Bernatova I, Pechanova O, Kristek F (1999) Mechanism of structural remodelling of the rat aorta during long-term NG-nitro-L-arginine methyl ester treatment. Japanese Journal of Pharmacology, 81, 99e106. Biscoe TJ (1971) Carotid body: structure and function. Physiological Reviews, 51, 437e495. Boudreau N, Rabinovitch M (1991) Developmentally-regulated changes in extracellular matrix in endothelial and smooth muscle cells in the ductus arteriosus may be related to intimal proliferation. Laboratory Investigation, 64, 187e199. Chiocchio SR, Biscardi AM, Tramezzani JH (1966) Catecholamines in the carotid body of the cat. Nature, 212, 834e835. Clarke JA, Daly MB, Marshall JM, Ead HW, Hennessy EM (2000) Quantitative studies of the vasculature of the carotid body in the chronically hypoxic rat. Brazilian Journal of Medical and Biological Research, 33, 331e340. Conde SV, Obeso A, Rigual R, Monteiro EC, Gonzalez C (2006) Function of the rat carotid body chemoreceptors in ageing. Journal of Neurochemistry, 99, 711e723.
355
Dai W, Chen H, Jiang J, Kong W, Wang Y (2010) Silencing MR-1 attenuates inflammatory damage in mice heart induced by AngII. Biochemical and Biophysical Research Communications, 391, 1573e1578. Dhillon DP, Barer GR, Walsh M (1984) The enlarged carotid body of the chronically hypoxic and chronically hypoxic and hypercapnic rat: a morphometric analysis. Quarterly Journal of Experimental Physiology, 69, 301e317. Evangelisti R, Bodo M, Caruso A, Becchetti E, Carinci P (1984) Extracellular glycosaminoglycans (GAG) released by chick embryonic fibroblasts. A possible involvement of surface receptors. Cell and Tissue Research, 238, 241e245. Eyzaguirre C (2005) Chemical and electric transmission in the carotid body chemoreceptor complex. Biological Research, 38, 341e345. Feng J, Chen BY, Cui LY (2008) Carotid body-mediated changes of sympathetic nerve and their relationships with hypertension. Chinese Medical Journal, 121, 1732e1735. Fernandes-Santos C, Mendonca LS, Mandarim-deLacerda CA (2006) Beneficial effects of angiotensin II AT1 blocker on cardiovascular adverse remodeling due to nitric oxide synthesis blockade. International Journal of Morphology, 24, 309e318. Ferrara N, Houck K, Jakeman L, Leung DW (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Reviews, 13, 18e32. Fidone S, Gonzalez C (1982) Catecholamine synthesis in rabbit carotid body in vitro. Journal of Physiology, 333, 69e79. Fidone S, Gonzalez C, Yoshizaki K (1982) Effects of hypoxia on catecholamine synthesis in rabbit carotid body in vitro. Journal of Physiology, 333, 81e91. Fink J, Fan NY, Rosenfeld L, Stier CT Jr. (1998) Contribution of endothelin to the acute pressor response of L-NAME in stroke-prone spontaneously hypertensive rats. Journal of Cardiovascular Pharmacology, 31, 618e622. Fletcher EC, Miller J, Schaaf JW, Fletcher JG (1987) Urinary catecholamines before and after tracheostomy in patients with obstructive sleep apnea and hypertension. Sleep, 10, 35e44. Fullmer HM (1960) A comparative histochemical study of elastic, pre-elastic and oxytalan connective tissue fibers. Journal of Histochemistry and Cytochemistry, 8, 290e295. Gkaliagkousi E, Passacquale G, Douma S, Zamboulis C, Ferro A (2010) Platelet activation in essential hypertension: implications for antiplatelet treatment. American Journal of Hypertension, 23, 229e236. Habeck JO, Huckstorf C, Moritz V (1987) A comparison of carotid body volumes in normotensive Wistar rats (NWR), Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Biomedica et Biochimica Acta, 46, 907e910. Hallberg D, Pernow B (1975) Effect of substance P on various vascular beds in the dog. Acta Physiologica Scandinavica, 93, 277e285. Hingtgen SD, Li Z, Kutschke W, Tian X, Sharma RV et al. (2010) Superoxide scavenging and AKT inhibition in the myocardium ameliorate pressure overload-induced
356
A.S. Felix et al.
NFkB activation and cardiac hypertrophy. Physiological Genomics. [Epub ahead of print]. Izal-Azcarate A, Belzunegui S, San Sebastian W, GarridoGil P, Vazquez-Claverie M et al. (2008) Immunohistochemical characterization of the rat carotid body. Respiratory Physiology and Neurobiology, 161, 95e99. Kameda Y (1996) Immunoelectron microscopic localization of vimentin in sustentacular cells of the carotid body and the adrenal medulla of guinea pigs. Journal of Histochemistry and Cytochemistry, 44, 1439e1449. Kasal DA, Neves MF, Oigman W, Mandarim-deLacerda CA (2008) Allopurinol attenuates L-NAME induced cardiomyopathy comparable to blockade of angiotensin receptor. Histology and Histopathology, 23, 1241e1248. Kusakabe T, Hayashida Y, Matsuda H, Gono Y, Powell FL et al. (1998) Hypoxic adaptation of the peptidergic innervation in the rat carotid body. Brain Research, 806, 165e174. Kusakabe T, Hirakawa H, Matsuda H, Kawakami T, Takenaka T et al. (2003) Peptidergic innervation in the rat carotid body after 2, 4, and 8 weeks of hypocapnic hypoxic exposure. Histology and Histopathology, 18, 409e418. Mayet J, Hughes A (2003) Cardiac and vascular pathophysiology in hypertension. Heart, 89, 1104e1109. Mazzone SB, Hinrichsen CF, Geraghty DP (1997) Substance P receptors in brain stem respiratory centers of the rat: regulation of NK1 receptors by hypoxia. Journal of Pharmacology and Experimental Therapeutics, 282, 1547e1556. Moncada S, Higgs EA (2006) The discovery of nitric oxide and its role in vascular biology. British Journal of Pharmacology, 147, 193e201. Morgan M, Pack RJ, Howe A (1975) Nerve endings in rat carotid body. Cell and Tissue Research, 157, 255e272. Murphy MB, Murray C, Shorten GD (2001) Fenoldopam: a selective peripheral dopamine-receptor agonist for the treatment of severe hypertension. New England Journal of Medicine, 345, 1548e1557. Nakanishi K, Mattson DL, Cowley AW Jr. (1995) Role of renal medullary blood flow in the development of L-NAME hypertension in rats. American Journal of Physiology, 268, 317e323. Park KH, Park WJ, Kim MK, Jung JH, Choi S et al. (2010) Noninvasive brachial-ankle pulse wave velocity in hypertensive patients with left ventricular hypertrophy. American Journal of Hypertension, 23, 269e274. Prabhakar NR, Peng YJ, Kumar GK, Nanduri J, Di Giulio C (2009) Long-term regulation of carotid body function: acclimatization and adaptation. Advances in Experimental Medicine and Biology, 648, 307e317. Puchtler H, Waldrop FS, Valentine LS (1973) Fluorescence microscopic distinction between elastin and collagen. Histochemie Histochemistry, 35, 17e30. Qin H, Ishiwata T, Wang R, Kudo M, Yokoyama M et al. (2000) Effects of extracellular matrix on phenotype modulation and MAPK transduction of rat aortic smooth muscle cells in vitro. Experimental and Molecular Pathology, 69, 79e90. Rigual R, Lopez-Lopez JR, Gonzalez C (1991) Release of dopamine and chemoreceptor discharge induced by low
pH and high PCO2 stimulation of the cat carotid body. Journal of Physiology, 433, 519e531. Shirahata M, Balbir A, Otsubo T, Fitzgerald RS (2007) Role of acetylcholine in neurotransmission of the carotid body. Respiratory Physiology and Neurobiology, 157, 93e105. Smith ML (2007) Sleep apnoea and hypertension: physiological bases for a causal relation. Experimental Physiology, 92, 19e20. Swislocki A, Eason T, Kaysen GA (1995) Oral administration of the nitric oxide biosynthesis inhibitor, N-nitro-Larginine methyl ester (L-NAME), causes hypertension, but not glucose intolerance or insulin resistance, in rats. American Journal of Hypertension, 8, 1009e1014. Timmers L, van Keulen JK, Hoefer IE, Meijs MF, van Middelaar B et al. (2009) Targeted deletion of nuclear factor kappaB p50 enhances cardiac remodeling and dysfunction following myocardial infarction. Circulation Research, 104, 699e706. Tipoe GL, Fung ML (2003) Expression of HIF-1alpha, VEGF and VEGF receptors in the carotid body of chronically hypoxic rat. Respiratory Physiology and Neurobiology, 138, 143e154. Torres RA (2009) Carotid body and sympathetic activation in heart failure: a story of sensors and sensitivity. Cardiovascular Research, 81, 633e634. Wang G, Zhang Z, Ayala C (2010) Hospitalization costs associated with hypertension as a secondary diagnosis among insured patients aged 18e64 years. American Journal of Hypertension, 23, 275e281. Wang ZZ, Dinger B, Fidone SJ, Stensaas LJ (1998) Changes in tyrosine hydroxylase and substance P immunoreactivity in the cat carotid body following chronic hypoxia and denervation. Neuroscience, 83, 1273e1281. Wang ZZ, Dinger BG, Stensaas LJ, Fidone SJ (1995) The role of nitric oxide in carotid chemoreception. Biological Signals, 4, 109e116. Wang ZZ, Stensaas LJ, Bredt DS, Dinger BG, Fidone SJ (1994) Mechanisms of carotid body inhibition. Advances in Experimental Medicine and Biology, 360, 229e235. Whiteman P (1973) The quantitative measurement of alcian blueeglycosaminoglycan complexes. Biochemical Journal, 131, 343e350. Wilkinson KD (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Seminars in Cell and Developmental Biology, 11, 141e148. Yamamoto Y, K€ onig P, Henrich M, Dedio J, Kummer W (2006) Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body. Cell and Tissue Research, 325, 3e11. Ye JS, Tipoe GL, Fung PC, Fung ML (2002) Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases. Pflugers Archiv, 444, 178e185.
February 18th, 2011 ½ Received, Accepted, July 25th, 2011
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EXPERIMENTALLY INDUCED DISEASE
Carotid Body Remodelling in L-NAME-Induced Hypertension in the Rat A. S. Felix, V. N. Rocha, A. L. R. Nascimento and J. J. de Carvalho Laboratory of Ultrastructure and Tissue Biology, Institute of Biology Roberto Alcantara Gomes, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Summary The carotid body (CB) is a chemoreceptor organ located at the bifurcation of the common carotid artery. It is made up of the carotid glomus, a structure containing type 1 cells surrounded by type 2 cells. The aim of this study was to evaluate the morphological changes of the CB and carotid glomus in the rat model of L-NAME-induced hypertension. Male Wistar rats were divided in two groups: control untreated rats (C) and rats receiving L-NAME 40 mg/kg/day (LN) for 6 weeks. At the end of the experiment, the systolic blood pressure was 63% higher in the LN group compared with the C group. Morphometric analysis showed that the area of the CB was 29% greater in the LN group compared with the C group. The density of nuclei in the CB was similar between groups, but it was 31% less in the carotid glomus of the LN group. Cells in the CB of the LN group displayed cytoplasmic vacuolation and expressed several biogenic amines. There were more elastic fibres, proteoglycans and collagen fibres in the LN group compared with the C group. Immunohistochemistry showed increased expression of nuclear factor kB, substance P, vascular endothelial growth factor and neuronal nitric oxide synthase in the LN group, while expression of the protein gene product 9.5 was decreased. L-NAME alters cell morphology and the expression of extracellular matrix molecules in the CB and carotid glomus in rats with L-NAME-induced hypertension. Ó 2011 Elsevier Ltd. All rights reserved. Keywords: carotid body; carotid glomus; hypertension; L-NAME
Introduction Systemic hypertension augments the risk of morbidity and mortality due to cardiovascular disease (Mayet and Hughes, 2003; Gkaliagkousi et al., 2010; Park et al., 2010; Wang et al., 2010). The endothelium plays a central role in regulating the vascular tone through the synthesis and release of vasoactive substances such as nitric oxide (NO), prostaglandin (PG) I2 and endothelium-derived hyperpolarizing factor (EDHF), as well as vasoconstrictor factors (Qin et al., 2000; Gkaliagkousi et al., 2010). Endothelial dysfunction in hypertension triggers an imbalance between the production and release of these factors, increasing the generation of reactive oxygen species and diminishing NO synthesis and bioavailability. L-arginine is the precursor of NO Correspondence to: J. J. de Carvalho (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2011.07.007
synthesis by NO synthase (NOS), an enzyme that exists in three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS) (Moncada and Higgs, 2006). Administration of L-arginine analogues such as L-NAME (hydrochloride NG-nitro-methylester-L-arginine) inhibits NOS activity and hence NO biosynthesis, leading to hypertension (Nakanishi et al., 1995; Swislocki et al., 1995; Fink et al., 1998; Bernatova et al., 1999; Fernandes-Santos et al., 2006). The carotid body (CB) is a chemoreceptor organ located at the bifurcation of the common carotid artery. The CB senses blood gas levels and regulates ventilation (Biscoe, 1971; Feng et al., 2008; Abudara and Eyzaguirre, 2008; Izal-Azcarate et al., 2008; Torres, 2009). It has a neural origin that can be characterized by the presence of the protein gene product 9.5 (PGP 9.5), a hydrolase found in neural and neuroendocrine tissues (Wilkinson, 2000). In rats, the CB consists of cellular aggregates (the carotid Ó 2011 Elsevier Ltd. All rights reserved.
Carotid Body Morphology in Hypertension
glomus) interspersed between blood vessels and nerve bundles. The carotid glomus has four to seven type 1 cells (or glomus cells) incompletely surrounded by two to three type 2 cells (or sustentacular cells) (Izal-Azcarate et al., 2008). Type 1 cells are characterized by the presence of numerous cytoplasmic vesicles containing dopamine, a biogenic amine, and gap junctions between adjacent cells (Morgan et al., 1975; Eyzaguirre, 2005). Afferent nerve endings establish synaptic contact with one or more type 1 cells (Rigual et al., 1991; Conde et al., 2006). Taquinin is an excitatory neurotransmitter that has a role in the brainstem-mediated response to CB stimulation. The tachykinin, P substance (PS) and the amino acid, L-glutamate, appear to be important excitatory neurotransmitters. PS acts on NK1 (PS receptor), a typical G protein-coupled receptor located in the afferent nerve endings of the CB (Mazzone et al., 1997). The extracellular matrix of the CB is composed of ground substance (i.e. glycosaminoglycans, proteoglycans and adhesive glycoproteins) and fibres (Evangelisti et al., 1984; Boudreau and Rabinovitch, 1991). However, data are scant regarding CB morphology in hypertension. It is known that there is a significant increase in CB volume in spontaneously hypertensive rats (SHRs) compared with normotensive Wistar Kyoto rats (Habeck et al., 1987). Similarly, New Zealand genetically hypertensive rats have fewer type 1 cells than control rats (Bee et al., 1989). Finally, abnormalities in dopamine production have been described in severe hypertension in man (Murphy et al., 2001). We hypothesize that systemic hypertension changes the normal morphology and protein expression of the CB. The aim of the present study was to characterize the morphological changes in the CB and carotid glomus in the rat model of L-NAME-induced hypertension.
Materials and Methods Animals and Sample collection
Three-month old Male Wistar rats were obtained from the State University of Rio de Janeiro (UERJ). Rats were kept under standard housing conditions (21 2 C, 60 10% humidity and a 12 h lightedark cycle) and received water and standard rodent chow (Nuvilab, Parana, Brazil) ad libitum. All procedures were approved by the Ethics and Research Committee of the Institute of Biology Roberto Alcantara Gomes/UERJ (Protocol 257/08) and were carried out in accordance with the conventional guidelines for the Care and Use of Laboratory Animals (US National Institutes of Health, revised 1996).
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Animals were divided into two groups (n ¼ 10): untreated rats (control group, C) or rats treated with L-NAME (SigmaeAldrich, St Louis, Missouri, USA) at a dosage of 40 mg/kg/day in their drinking water (L-NAME group, LN) (Bernatova et al., 1999; Kasal et al., 2008). Body mass and systolic blood pressure (SBP) were measured weekly throughout the 6 weeks of the experiment by plethysmographic flow (LETICA LE 5100; Panlab, Barcelona, Spain). At the end of the experiment, rats were anaesthetized with sodium pentobarbital (50 mg/kg by intraperitoneal injection). An incision was made in the neck to expose the carotid bifurcation and then the CB was rapidly freed from adherent adipose and connective tissue for isolation and fixation. Samples were collected from both sides of the neck and processed for light (n ¼ 6) and electron (n ¼ 4) microscopy. Microscopy
CB samples were fixed in 4% paraformaldehyde in 5 mM CaCl2 and in 0.1% glutaraldehyde and were processed routinely and embedded in paraplast plus (SigmaeAldrich). Sections (3 mm) were stained with haematoxylin and eosin (HE), Weigert’s resorcinfuchsin (Fullmer, 1960), picrosirius red (to demonstrate collagen fibres) (Puchtler et al., 1973) and alcian blue pH 2.5 (to demonstrate proteoglycans) (Whiteman, 1973). In order to study the vesicles containing biological amines, CB samples were examined with a laser scanning confocal microscope (LSM 510 Meta; Carl Zeiss, G€ottingen, Germany) using formaldehyde-induced fluorescence (FIF).
Immunohistochemistry
CB sections were incubated with primary antibodies specific for: vascular endothelial growth factor (VEGF; dilution 1 in 100; Santa Cruz Biotechnology, Santa Cruz, California, USA; sc-7269), nuclear factor kB (NF-kB; dilution 1 in 100; Santa Cruz Biotechnology; sc-114), substance P (PS; dilution 1 in 100; Chemicon, Billerica, Massachusetts, USA; AB1566), nNOS (dilution 1 in 50, Santa Cruz Biotechnology; sc-648) and PGP 9.5 (dilution 1 in 50; Abcam; AB8189). The reaction was amplified using a biotinestreptavidin complex system (LSAB Kit Universal; DakoCytomation, Glostrup, Denmark) and the immunolabelling was ‘visualized’ after incubation with 3, 30 diaminobenzidine tetrachloride (DAB; DakoCytomation). Sections were counterstained with Mayer’s haematoxylin.
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Electron Microscopy
CB samples fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and 0.25% tannic acid (Merck, Darmstadt, Germany) were post-fixed in osmium tetroxide 1% (SigmaeAldrich) and potassium ferrocyanide 0.8% in 0.1 M cacodylate buffer (pH 7.2) for 1 h and then dehydrated in acetone and embedded in Epon (Embed-812; Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). Semithin sections (1 mm) were stained with toluidine blue and observed by light microscopy to select the areas of interest. Ultrathin sections (Leica Ultracut-UCT, Vienna, Austria) were contrasted with uranyl acetate and lead citrate and analyzed with a transmission electron microscope (Zeiss EM 906; Carl Zeiss, Oberkochen, Germany) at 80 kV. CB sections were fixed in paraformaldehyde 4%, processed and embedded in LR-White resin for immunoelectronmicroscopical analysis of expression of the enzyme tyrosine hydroxylase (TH). The secondary antibodies were conjugated to colloidal gold particles (10 nm). After immunolabelling, the sections were contrasted with uranyl acetate and lead citrate and analyzed by transmission electron microscopy (TEM) as above.
Fig. 1. Selection of the CB area chosen for measurement. Measurement was performed by the functions ‘clear outside’ and ‘measure’ of the Image J software.
the Student’s t-test. P # 0.05 was considered statistically significant (GraphPad Prism version 5.03, San Diego, California, USA).
Morphometry
Digital images were obtained using a digital camera (Olympus DP70, Tokyo, Japan) attached to a microscope (Olympus BX51). Sections stained with HE were used to quantify the density of nuclei in the CB and carotid glomus. Firstly, the boundary of the CB was outlined and its area was assessed (Image J software version 1.4, National Institutes of Health, Bethesda, Maryland, USA). Then, all nuclei (type 1 and type 2 cells) located inside the carotid glomus were quantified. The density of nuclei per CB (nuclei/mm2 of CB area) was calculated as: number of nuclei/image area (Fig. 1). Secondly, to quantify the density of nuclei in the carotid glomus, its boundary was first outlined and the area calculated. Then, the number of nuclei inside the carotid glomus was automatically calculated by the software. The density of nuclei per carotid glomus (nuclei/mm2 of carotid glomus area) was calculated as: number of cells inside the carotid glomus/carotid glomus area. Seven rats were analyzed per group and for each sample, 20 fields were examined, totalling 140 fields per group.
Results L-NAME increased the SBP and induced body weight loss in the LN group compared with the C group (Table 1). A difference in SBP was first noticed in the second week of the experiment and it increased in the following weeks. SBP reached 190 7.01 mmHg in the LN group compared with 122.2 2.51 mmHg in the C group (an increase of 63%). Body weight was similar between C and LN groups at the beginning
Table 1 Blood pressure, body mass and CB morphometry Control
L-NAME
P
117 5.8 190 7.01
NS 0.0001
265.4 13.0 306.0 14.9 152,400 12,030 5,877 361 4,936 378
NS 0.0002 0.0056 NS 0.0003
Statistics
Blood pressure (mm Hg) Initial 120 7.0 Final 122.2 2.51 Body mass (g) Initial 271.3 9.0 Final 375.6 24.2 106,000 7,181 CB area (mm2) 6,602 160 Nuclei/CB (mm2) 7,190 350 Nuclei/carotid glomus (mm2)
Data are shown as mean and standard error of the mean (SEM). Data were tested for normality. Differences between the C and LN groups were analyzed by
Data are shown as mean SEM, Student’s t-test. Initial refers to data assessed before the beginning of the experiment (week 0) and final are data on week 6. NS, non-significant.
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of the experiment, but it had decreased by 19% in LN group in the 6th week compared with the C group. The CB area increased in the LN group by 29% compared with the C group (Table 1). The density of nuclei in the CB was similar between the C and LN groups, suggesting CB cell hypertrophy rather than hyperplasia. Additionally, the density of nuclei in the carotid glomus was 31% less in the LN group compared with the C group. L-NAME-induced morphological changes in the carotid glomus are shown in Fig. 1. In the LN group, the regular morphology of the CB was lost as the carotid glomus cells were hypertrophic and had cytoplasmic vacuolation (Figs. 2a, b). The FIF technique allows the visualization of biogenic amines, and there was a greater amount of these in the LN group compared with the C group (Figs. 2c, d). Some electron-dense granules were
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found in the cytoplasm of cells of the carotid glomus in the C group, but these were more frequently seen in the LN group (Figs. 2e, f). The presence of vacuoles in the cytoplasm of the carotid glomus cells (Figs. 2e, f) was also evident by TEM. TEM analysis for expression of TH showed reduced expression of the enzyme in the cytoplasm of type 1 cells in the LN group compared with those cells in the C group. Colloidal gold particles were found in smaller numbers in the hypertensive group (LN) (Fig. 3). Morphological changes in the extracellular matrix are depicted in Fig. 4. The LN group displayed more elastic fibres (Figs. 4a, b) and proteoglycans (Figs. 4c, d) compared with the C group. The picrosirius red stain, without (Figs. 4e, f) or with (Figs. 4g, h) light polarization, revealed an increased number of collagen fibres in the LN group compared with the C group.
Fig. 2. CB structure and ultrastructure. (a, b) Photomicrographs. HE. Bar, 30 mm. (c, d) FIF; arrows indicate biogenic amines. Bar, 30 mm. (e, f) TEM of the CB. Bar, 2 mm. White arrows indicate the biogenic amines in the CB. Oval white arrows show the location of biogenic amines in cells of the carotid glomus.
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Fig. 3. Immunoelectronmicroscopy of the CB with primary anti-TH antibody and a secondary antibody conjugated to 10 nm colloidal gold particles. White arrows indicate the labelling in the cytoplasm of the CB type 1 cells. (a, b) Group C. (c, d) LN group.
The expression of five different proteins was analyzed by immunohistochemistry. L-NAME treatment increased the expression of NF-kB, substance P, VEGF and nNOS compared with the C group. PGP 9.5 was the only protein that had its expression diminished by L-NAME treatment. NF-kB was expressed in the extracellular matrix, while substance P and PGP 9.5 were found in the carotid glomus. VEGF was mainly expressed by glomus cells in the C group, but it was distributed in scattered fashion throughout the cytoplasm of glomus cells and blood vessels in the LN group. Finally, nNOS was more highly expressed in the carotid glomus and extracellular matrix in the LN group, a pattern not seen in the C group (Fig. 5).
Discussion The NOS inhibitor L-NAME has been used extensively as a means of inducing hypertension in animal models (Kasal et al., 2008). The results of the
present study have shown that L-NAME-induced hypertension alters the amount of extracellular matrix proteins and cellular morphology of the CB. The increase in elastic fibres, proteoglycans and collagen fibres correlates with the increased CB area in L-NAME-treated rats. Changes in protein expression are thought to reflect the increased blood pressure and sympathetic activity and probably occur in order to normalize the physiological function of the CB. CB volume increases in rats subjected to chronic hypoxia (Dhillon et al., 1984; Clarke et al., 2000) and the same phenomenon is observed in New Zealand genetically hypertensive rats (Bee et al., 1989). An enlargement of the CB was also noticed in the present study. This was due to a combination of enhancement of extracellular matrix deposition and hypertrophy of the carotid glomus cells. The extracellular matrix deposition was characterized as an increased amount of elastic fibres, proteoglycans and collagen. The density of nuclei in the carotid glomus was diminished, while the carotid
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Fig. 4. Photomicrographs showing the extracellular matrix of the CB. (a, b) Weigert’s resorcin-fuchsin stain. (c, d) Alcian blue stain. (e, f) Picrosirius red stain. (g, h) Picrosirius red stain under polarized light. Bars, 30 mm. Thin black arrows indicate the presence of elastic fibres around blood vessels. Oval black arrows indicate the presence of elastic fibres within the extracellular matrix of the CB. Diamond black arrows indicate the deposition of proteoglycans around blood vessels. White arrowheads indicate collagen fibres within the CB.
area was bigger, suggesting hypertrophy of the carotid glomus cells. NF-kB is a protein complex expressed in almost all animal cells and it controls DNA transcription. In the heart, NF-kB activation enhances cardiac remodelling and dysfunction following myocardial infarction (Timmers et al., 2009), angiotensin II-induced cardiac hypertrophy (Dai et al., 2010) and pressure overload-induced left ventricular hypertrophy (Hingtgen et al., 2010). We found increased expression of NF-kB in L-NAME-treated rats, thus this complex may be driving both extracellular matrix remodelling and the hypertrophy of carotid glomus cells. PGP 9.5 has been described in glomus type 1 cells (Kameda, 1996) and its expression in the CB was diminished by L-NAME treatment. This result is probably due to a reduction in the deubiquitination process and thus accumulation of unnecessary proteins for CB cellular metabolism. L-NAME-treated rats had greater amounts of biogenic amines within the CB. The observed electron-dense granules likely represent biogenic amines, as does the fluorescence depicted in Figs. 1c, d. Biogenic amines such as catecholamines are particu-
larly found in vesicles located in the cytoplasm of type 1 cells of the glomus (Shirahata et al., 2007). Studies of patients with obstructive sleep apnoea and cardiovascular morbidity, particularly systemic hypertension, show a significant increase in the production and circulation of catecholamines during apnoea (Fletcher et al., 1987; Smith, 2007). This neurotransmitter is released and increases sympathetic activity in order to maintain blood pressure during sleep disorders and hypoxia (Chiocchio et al., 1966; Fidone et al., 1982). Glomus type 1 cells also produce the excitatory neurotransmitter SP. During hypoxia there is an increased demand, suggesting that nerves containing the SP receptor are involved in the chemosensory response and vascular dilation (Hallberg and Pernow, 1975; Kusakabe et al., 1998, 2003). In the present study, rats subjected to NOS inhibition had increased expression of SP, suggesting that this molecule is being used as a chemosensory stimulus and thus might be an important vasodilator to improve blood circulation and keep oxygen reaching the tissues. Dopamine, a catecholamine, is produced from the non-essential amino acid tyrosine and is transformed into L-DOPA by TH. Type 1 cells have been
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Fig. 5. Immunohistochemical analysis of the CB. (a, b) NF-kB. (c, d) PS. (e, f) protein PGP 9.5. (g, h) VEGF. (i, j) nNOS. (k, l) Negative control. Bars, 30 mm.
identified, in previous studies, as the main storage site for catecholamines in the CB. Dopamine is probably a neurotransmitter of afferent fibre discharge (Fidone and Gonzalez, 1982). The release of this neurotransmitter might alter stimulatory capacity of type 1 cells (Wang et al., 1998). Experimental studies have shown that levels of dopamine and noradrenaline decrease after an episode of acute hypoxia, suggesting that these molecules are released by type 1 cells (Rigual et al., 1991) in order to normalize the levels of blood oxygenation. The results of the present study show a reduction in TH in the hypertensive group (LN) compared with the control group (C) in response to L-NAME, even though there was an increase in catecholamines in the CB of the LN group. An explanation for this finding would be that the hypertension induced by L-NAME increases the production and storage of these biogenic amines within
the cytoplasm of the type 1 cells before the remodelling of the organ, with loss of their structure and reduction in TH level. Available data indicate that NOS isoforms produce NO within diverse cell types in the CB, such as the vascular endothelial cells, parasympathetic neurons, afferent nerve terminals and glomus type 1 cells (Ye et al., 2002; Yamamoto et al., 2006). The neuronal isoform of NOS is contained in a dense plexus of afferent nerve fibres that terminate near oxygen-sensitive type 1 cells in the CB of rats. Additionally, a distinct group of nNOS-positive parasympathetic neurons have been shown to innervate the CB vasculature (Wang et al., 1994, 1995). In the present study, there was increased expression of nNOS in glomus cells and in components of the extracellular matrix of LNAME-treated rats. VEGF increases vascularization and restores oxygen supply when blood flow to
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tissues becomes inadequate (Ferrara et al., 1992; Prabhakar et al., 2009). VEGF expression in CB also increased following inhibition of NOS and a similar result was found by Tipoe and Fung (2003) in rats exposed to chronic hypoxia. Hence, the increased expression of nNOS and VEGF in L-NAME-treated rats indicates an attempt to increase the synthesis of the vasodilator NO and tissue perfusion in the CB. In conclusion, the present study has shown that induction of hypertension in rats treated with L-NAME changes cellular morphology and the expression of extracellular matrix proteins in the CB and carotid glomus.
Acknowledgments The authors are grateful to the Brazilian Council of Science and Technology (CNPq), the Rio de Janeiro State Foundation for Scientific Research (FAPERJ), the Council for the Improvement of Graduate Personnel (CAPES) and the State University of Rio de Janeiro (UERJ).
Conflict of Interests The authors disclose no conflict of interest.
References Abudara V, Eyzaguirre C (2008) Mechanical sensitivity of carotid body glomus cells. Respiratory Physiology and Neurobiology, 161, 210e213. Bee D, Barer G, Wach R, Pallot D, Emery C et al. (1989) Structure and function of the carotid body in New Zealand genetically hypertensive rats. Quarterly Journal of Experimental Physiology, 74, 691e701. Bernatova I, Pechanova O, Kristek F (1999) Mechanism of structural remodelling of the rat aorta during long-term NG-nitro-L-arginine methyl ester treatment. Japanese Journal of Pharmacology, 81, 99e106. Biscoe TJ (1971) Carotid body: structure and function. Physiological Reviews, 51, 437e495. Boudreau N, Rabinovitch M (1991) Developmentally-regulated changes in extracellular matrix in endothelial and smooth muscle cells in the ductus arteriosus may be related to intimal proliferation. Laboratory Investigation, 64, 187e199. Chiocchio SR, Biscardi AM, Tramezzani JH (1966) Catecholamines in the carotid body of the cat. Nature, 212, 834e835. Clarke JA, Daly MB, Marshall JM, Ead HW, Hennessy EM (2000) Quantitative studies of the vasculature of the carotid body in the chronically hypoxic rat. Brazilian Journal of Medical and Biological Research, 33, 331e340. Conde SV, Obeso A, Rigual R, Monteiro EC, Gonzalez C (2006) Function of the rat carotid body chemoreceptors in ageing. Journal of Neurochemistry, 99, 711e723.
355
Dai W, Chen H, Jiang J, Kong W, Wang Y (2010) Silencing MR-1 attenuates inflammatory damage in mice heart induced by AngII. Biochemical and Biophysical Research Communications, 391, 1573e1578. Dhillon DP, Barer GR, Walsh M (1984) The enlarged carotid body of the chronically hypoxic and chronically hypoxic and hypercapnic rat: a morphometric analysis. Quarterly Journal of Experimental Physiology, 69, 301e317. Evangelisti R, Bodo M, Caruso A, Becchetti E, Carinci P (1984) Extracellular glycosaminoglycans (GAG) released by chick embryonic fibroblasts. A possible involvement of surface receptors. Cell and Tissue Research, 238, 241e245. Eyzaguirre C (2005) Chemical and electric transmission in the carotid body chemoreceptor complex. Biological Research, 38, 341e345. Feng J, Chen BY, Cui LY (2008) Carotid body-mediated changes of sympathetic nerve and their relationships with hypertension. Chinese Medical Journal, 121, 1732e1735. Fernandes-Santos C, Mendonca LS, Mandarim-deLacerda CA (2006) Beneficial effects of angiotensin II AT1 blocker on cardiovascular adverse remodeling due to nitric oxide synthesis blockade. International Journal of Morphology, 24, 309e318. Ferrara N, Houck K, Jakeman L, Leung DW (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocrine Reviews, 13, 18e32. Fidone S, Gonzalez C (1982) Catecholamine synthesis in rabbit carotid body in vitro. Journal of Physiology, 333, 69e79. Fidone S, Gonzalez C, Yoshizaki K (1982) Effects of hypoxia on catecholamine synthesis in rabbit carotid body in vitro. Journal of Physiology, 333, 81e91. Fink J, Fan NY, Rosenfeld L, Stier CT Jr. (1998) Contribution of endothelin to the acute pressor response of L-NAME in stroke-prone spontaneously hypertensive rats. Journal of Cardiovascular Pharmacology, 31, 618e622. Fletcher EC, Miller J, Schaaf JW, Fletcher JG (1987) Urinary catecholamines before and after tracheostomy in patients with obstructive sleep apnea and hypertension. Sleep, 10, 35e44. Fullmer HM (1960) A comparative histochemical study of elastic, pre-elastic and oxytalan connective tissue fibers. Journal of Histochemistry and Cytochemistry, 8, 290e295. Gkaliagkousi E, Passacquale G, Douma S, Zamboulis C, Ferro A (2010) Platelet activation in essential hypertension: implications for antiplatelet treatment. American Journal of Hypertension, 23, 229e236. Habeck JO, Huckstorf C, Moritz V (1987) A comparison of carotid body volumes in normotensive Wistar rats (NWR), Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Biomedica et Biochimica Acta, 46, 907e910. Hallberg D, Pernow B (1975) Effect of substance P on various vascular beds in the dog. Acta Physiologica Scandinavica, 93, 277e285. Hingtgen SD, Li Z, Kutschke W, Tian X, Sharma RV et al. (2010) Superoxide scavenging and AKT inhibition in the myocardium ameliorate pressure overload-induced
356
A.S. Felix et al.
NFkB activation and cardiac hypertrophy. Physiological Genomics. [Epub ahead of print]. Izal-Azcarate A, Belzunegui S, San Sebastian W, GarridoGil P, Vazquez-Claverie M et al. (2008) Immunohistochemical characterization of the rat carotid body. Respiratory Physiology and Neurobiology, 161, 95e99. Kameda Y (1996) Immunoelectron microscopic localization of vimentin in sustentacular cells of the carotid body and the adrenal medulla of guinea pigs. Journal of Histochemistry and Cytochemistry, 44, 1439e1449. Kasal DA, Neves MF, Oigman W, Mandarim-deLacerda CA (2008) Allopurinol attenuates L-NAME induced cardiomyopathy comparable to blockade of angiotensin receptor. Histology and Histopathology, 23, 1241e1248. Kusakabe T, Hayashida Y, Matsuda H, Gono Y, Powell FL et al. (1998) Hypoxic adaptation of the peptidergic innervation in the rat carotid body. Brain Research, 806, 165e174. Kusakabe T, Hirakawa H, Matsuda H, Kawakami T, Takenaka T et al. (2003) Peptidergic innervation in the rat carotid body after 2, 4, and 8 weeks of hypocapnic hypoxic exposure. Histology and Histopathology, 18, 409e418. Mayet J, Hughes A (2003) Cardiac and vascular pathophysiology in hypertension. Heart, 89, 1104e1109. Mazzone SB, Hinrichsen CF, Geraghty DP (1997) Substance P receptors in brain stem respiratory centers of the rat: regulation of NK1 receptors by hypoxia. Journal of Pharmacology and Experimental Therapeutics, 282, 1547e1556. Moncada S, Higgs EA (2006) The discovery of nitric oxide and its role in vascular biology. British Journal of Pharmacology, 147, 193e201. Morgan M, Pack RJ, Howe A (1975) Nerve endings in rat carotid body. Cell and Tissue Research, 157, 255e272. Murphy MB, Murray C, Shorten GD (2001) Fenoldopam: a selective peripheral dopamine-receptor agonist for the treatment of severe hypertension. New England Journal of Medicine, 345, 1548e1557. Nakanishi K, Mattson DL, Cowley AW Jr. (1995) Role of renal medullary blood flow in the development of L-NAME hypertension in rats. American Journal of Physiology, 268, 317e323. Park KH, Park WJ, Kim MK, Jung JH, Choi S et al. (2010) Noninvasive brachial-ankle pulse wave velocity in hypertensive patients with left ventricular hypertrophy. American Journal of Hypertension, 23, 269e274. Prabhakar NR, Peng YJ, Kumar GK, Nanduri J, Di Giulio C (2009) Long-term regulation of carotid body function: acclimatization and adaptation. Advances in Experimental Medicine and Biology, 648, 307e317. Puchtler H, Waldrop FS, Valentine LS (1973) Fluorescence microscopic distinction between elastin and collagen. Histochemie Histochemistry, 35, 17e30. Qin H, Ishiwata T, Wang R, Kudo M, Yokoyama M et al. (2000) Effects of extracellular matrix on phenotype modulation and MAPK transduction of rat aortic smooth muscle cells in vitro. Experimental and Molecular Pathology, 69, 79e90. Rigual R, Lopez-Lopez JR, Gonzalez C (1991) Release of dopamine and chemoreceptor discharge induced by low
pH and high PCO2 stimulation of the cat carotid body. Journal of Physiology, 433, 519e531. Shirahata M, Balbir A, Otsubo T, Fitzgerald RS (2007) Role of acetylcholine in neurotransmission of the carotid body. Respiratory Physiology and Neurobiology, 157, 93e105. Smith ML (2007) Sleep apnoea and hypertension: physiological bases for a causal relation. Experimental Physiology, 92, 19e20. Swislocki A, Eason T, Kaysen GA (1995) Oral administration of the nitric oxide biosynthesis inhibitor, N-nitro-Larginine methyl ester (L-NAME), causes hypertension, but not glucose intolerance or insulin resistance, in rats. American Journal of Hypertension, 8, 1009e1014. Timmers L, van Keulen JK, Hoefer IE, Meijs MF, van Middelaar B et al. (2009) Targeted deletion of nuclear factor kappaB p50 enhances cardiac remodeling and dysfunction following myocardial infarction. Circulation Research, 104, 699e706. Tipoe GL, Fung ML (2003) Expression of HIF-1alpha, VEGF and VEGF receptors in the carotid body of chronically hypoxic rat. Respiratory Physiology and Neurobiology, 138, 143e154. Torres RA (2009) Carotid body and sympathetic activation in heart failure: a story of sensors and sensitivity. Cardiovascular Research, 81, 633e634. Wang G, Zhang Z, Ayala C (2010) Hospitalization costs associated with hypertension as a secondary diagnosis among insured patients aged 18e64 years. American Journal of Hypertension, 23, 275e281. Wang ZZ, Dinger B, Fidone SJ, Stensaas LJ (1998) Changes in tyrosine hydroxylase and substance P immunoreactivity in the cat carotid body following chronic hypoxia and denervation. Neuroscience, 83, 1273e1281. Wang ZZ, Dinger BG, Stensaas LJ, Fidone SJ (1995) The role of nitric oxide in carotid chemoreception. Biological Signals, 4, 109e116. Wang ZZ, Stensaas LJ, Bredt DS, Dinger BG, Fidone SJ (1994) Mechanisms of carotid body inhibition. Advances in Experimental Medicine and Biology, 360, 229e235. Whiteman P (1973) The quantitative measurement of alcian blueeglycosaminoglycan complexes. Biochemical Journal, 131, 343e350. Wilkinson KD (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Seminars in Cell and Developmental Biology, 11, 141e148. Yamamoto Y, K€ onig P, Henrich M, Dedio J, Kummer W (2006) Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body. Cell and Tissue Research, 325, 3e11. Ye JS, Tipoe GL, Fung PC, Fung ML (2002) Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases. Pflugers Archiv, 444, 178e185.
February 18th, 2011 ½ Received, Accepted, July 25th, 2011