A locally generated angiotensin system in rat carotid body

doi:10.1016/S0167-0115(02)00068-X

Regulatory Peptides

Volume 107, Issues 1–3, 15 July 2002, Pages 97-103

https://doi.org/10.1016/S0167-0115(02)00068-X Get rights and content

Abstract

Previous studies have shown the existence of functional angiotensin II receptors in rat carotid body, which directly alters the carotid chemoreceptor afferent nerve activity. Moreover, chronic hypoxia could result in an enhanced sensitivity of chemoreceptor afferent activity via an AT₁ receptor-mediated calcium signaling in the carotid body. In the present study, the localization and expression of angiotensinogen, the obligatory component for an intrinsic, angiotensin-generating system, were investigated by in situ hybridization histochemistry, immunohistochemistry, RT-PCR, Western blot and Northern blot analysis. In situ hybridization showed the expression of angiotensinogen within the glomus cells of the carotid body. Double immunostaining of angiotensinogen and tyrosine hydroxylase, an immunohistochemical marker for type I glomus cells, elucidated that angiotensinogen protein was specifically localized to the lobules of type I cells. Consistently, RT-PCR and Western blot analysis confirmed the expression of angiotensinogen mRNA and protein, respectively. On the other hand, renin mRNA was not detected using RT-PCR and Northern blot analysis whereas angiotensin-converting enzyme (ACE) mRNA was detected in the carotid body. These data suggest that a locally generated angiotensin system is operated in the carotid body, which might be linked to a renin-independent biosynthetic pathway. Such an intrinsic, angiotensin-generating system and its local regulation by chronic hypoxia should be important in the modulation of cardiopulmonary adaptation in the hypoxic ventilatory response and the electrolyte as well as water homeostasis.

Introduction

The carotid body is a highly vascularized network of chemoreceptor cells, which are located at the bifurcation of the internal and external carotid arteries arising from the common carotid artery. Carotid body chemoreceptors, called type I glomus cells, sense PaO₂, PaCO₂ and pH, and release neurotransmitters. These in turn determine the action potential frequency in the sensory fibers of the carotid sinus nerve [1]. Evidence has shown that type I cells are the receptor cells for peripheral chemoreception and their activity is associated with the intracellular calcium in carotid body [2], [3], [4].

The physiological function of carotid body is vital for arterial oxygen tension [5], [6] and it can be excited by hypoxia [7]. In hypoxia, carotid body chemoreceptors increase the afferent activity of the carotid sinus nerves, and thereby enhancing the brainstem activities for the elevation of the respiratory drives [8]. The activation of chemoreceptors then increases reflexly the cardiorespiratory functions. It also results in the secretion of vasoactive hormones including noradrenaline and angiotensin II and thus further stimulating the cardiovascular actions [9].

Angiotensin II has long been known to be a vasoconstrictor peptide in the maintenance of blood pressure and anion as well as fluid homeostasis [10]. The physiologically active peptide is derived from its hepatic angiotensinogen by a sequential action of two critical enzymes [11]. The first renal renin cleaves the precursor angiotensinogen into angiotensin I, which is then split to angiotensin II by the second pulmonary ACE. Circulating angiotensin II can exert diverse physiological functions via the specific interactions with its membrane-bound receptors such as AT₁ and AT₂ receptors [12].

In the carotid body, angiotensin II has been previously shown to increase the chemoreceptor afferent activity, presumably via the interaction with AT₁ receptor in the type I glomus cells [13]. Our recent study has further demonstrated the functional expression of AT₁ receptor, which could mediate the intracellular calcium release by the carotid body chemoreceptors [14]. Moreover, chronic hypoxia can upregulate the AT₁ receptor, resulting in an increase of carotid body afferent nerve activity to angiotensin II stimulation [15]. In spite of the close association between the regulation and function of AT₁ receptor in the carotid body, there has been no evidence for the existence of an intrinsic, angiotensin-generating system in the carotid body. The present study aims at elaborating the expression and localization of angiotensinogen, which is the mandatory component for a locally generated angiotensin system, in the carotid body.

Section snippets

Experimental animals and isolation of carotid body

Adult Sprague–Dawley (SD) rats aged 28 days were used. The animals were bred and raised under a pathogen-free condition with a controlled ambient temperature of 20±1 °C, relatively humidity of 60–80% and a 14-h light/10-h dark cycle in the Laboratory Animal Services Centre of the Chinese University of Hong Kong. Standard rat chow and tap water were supplied ad libitum. Ethical approval for the experimental procedures was obtained from the Animal Ethical Committee of the Chinese University of

Expression and localization of angiotensinogen mRNA

As a first step to identify the presence of angiotensinogen mRNA, the mandatory component for an intrinsic angiotensin system, RT-PCR was performed to determine its existence in the carotid body. Results from RT-PCR showed that angiotensinogen mRNA was expressed in the rat carotid body, although its expression level appeared to be low when compared with that in the liver (Fig. 1). On the other hand, the precise cellular localization and expression of angiotensinogen mRNA was further elucidated

Discussion

In the present study, the expression and localization of angiotensinogen, the indispensable component for the existence of a locally generated angiotensin system, were unequivocally elucidated in the rat carotid body. RT-PCR showed that rat carotid body could express mRNA of angiotensinogen. In situ hybridization demonstrated the precise localization of angiotensinogen mRNA to the clusters of glomus cells in the carotid body. Consistently, Western blot analysis and immunohistochemistry

Acknowledgements

We would like to gratefully acknowledge the financial support by the Competitive Earmarked Research Grant from the Research Grants Council of Hong Kong (Project No: CUHK 4075/00M and CUHK 4116/01M) and by the Direct Grant from the Chinese University of Hong Kong.

References (36)

P.S. Leung et al.
Angiotensinogen expression by rat epididymis: evidence for an intrinsic, angiotensin-generating system
Mol. Cell. Endocrinol.
(1999)
P.S. Leung et al.
Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis
Regul. Pept.
(2001)
P. Chomczynski et al.
Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987)
W.G. Thomas et al.
Immunocytochemical localization of angiotensinogen in the rat brain
Neuroscience
(1988)
P.S. Leung et al.
Testicular hormonal regulation of the renin–angiotensin system in the rat epididymis
Life Sci.
(2000)
P.S. Leung et al.
Angiotensin II receptors, AT₁ and AT₂ in the rat epididymis: immunocytochemical and electrophysiological studies
Biochim. Biophys. Acta
(1997)
T.J. Biscoe
Carotid body: structure and function
Physiol. Rev.
(1971)
I. Vicario et al.
Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception
Am. J. Physiol.
(2000)
K.J. Buckler
Role of potassium channels in hypoxic chemoreception in rat carotid body type-I cells
Adv. Exp. Med. Biol.
(1996)
C. Eyzaguirre et al.
Possible role of coupling between glomus cells in carotid body chemoreception
Biol. Signals
(1995)

D.F. Donnelly et al.

Hypoxia decreases intracellular calcium in adult rat carotid body cells

J. Neurophysiol.

(1992)

D.F. Donnelly

Developmental aspects of oxygen sensing by the carotid body

J. Appl. Physiol.

(2000)

L. He et al.

Cellular mechanisms involved in carotid body inhibition produced by atrial natriuretic peptide

Am. J. Physiol.

(2000)

C. Gonzalez et al.

Carotid body chemoreceptors: from natural stimuli to sensory discharges

Physiol. Rev.

(1994)

J.M. Marshall

Peripheral chemoreceptors and cardiovascular regulation

Physiol. Rev.

(1994)

M.J. Peach

Renin–angiotensin system: biochemistry and mechanisms of action

Physiol. Rev.

(1977)

E. Hackenthal et al.

Morphology, physiology, and molecular biology of renin secretion

Physiol. Rev.

(1990)

M. De Gasparo et al.

The angiotensin II receptors

Pharmacol. Rev.

(2000)

Cited by (60)

The contribution of angiotensin peptides to cardiovascular neuroregulation in health and disease
2023, Angiotensin: From the Kidney to Coronavirus
The brain renin–angiotensin system (RAS) is critically involved in the cardiovascular regulation and energy homeostasis. The overwhelming evidence indicates that the central RAS closely cooperates with the systemic RAS by means of interactions with the autonomic nervous system and hormonal and humoral factors linking RAS with regulation of blood pressure, water–electrolyte equilibrium, and energy balance. Particularly significant roles in these interactions are played by aldosterone and vasopressin; however, other factors such as proinflammatory cytokines, growth factors, nitric oxide, prostaglandins, and ROS appear to be also essential players, especially during challenges (stress, hypoxia), and under pathological conditions (hypertension, heart failure, metabolic syndrome, atherosclerosis, COVID-19). Current pharmacotherapies of cardiovascular diseases are focused on the angiotensin-converting enzyme inhibitors and angiotensin type 1 receptor blockers and their effects on systemic RAS. A better understanding of neuroanatomical and neurochemical connections between central RAS and other regulatory systems should advance pharmacological interventions aimed at central RAS.
Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats
2020, Acta Histochemica
The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.
Immunohistochemical localization of angiotensin AT <inf>1</inf> receptors in the rat carotid body
2018, Acta Histochemica
Citation Excerpt :
Ang II and its receptors are major components of the renin-angiotensin system (RAS). There is now compelling evidence that under physiological conditions a locally generated RAS exists in the rat CB, and that this system plays a key role in its modulatory response to hypoxia (Lam and Leung, 2002; Leung et al., 2003). More recently, it has been shown that multiple intermediate metabolites of this system and their corresponding receptors can influence the glomus cell function and enhance afferent chemoreceptor activity (Fung, 2015).
The carotid body (CB) is a major peripheral arterial chemoreceptor that initiates respiratory and cardiovascular adjustments to maintain homeostasis. Recent evidence suggests that circulating or locally produced hormones like angiotensin II acting via AT₁ receptors modulate its activity in a paracrine-autocrine manner. The aim of this study was to examine the immunohistochemical localization of AT₁ receptor in the CB of adult rats and to compare its expression in vehicle-treated animals, and after the long-term application of its selective blocker losartan. Immunohistochemistry revealed that a subset of CB glomeruli and the vast majority of neurons in the adjacent superior cervical ganglion (SCG) were strongly AT₁ receptor-immunoreactive. In the CB immunostaining was observed in the chemosensory glomus cells typically aggregated in cell clusters while the nerve fibers in-between and large capillaries around them were immunonegative. Exogenous administration of losartan for a prolonged time significantly reduces the intensity of AT₁ receptor immunostaining in the CB glomus cells and SCG neurons. Our results show that AT₁ receptors are largely expressed in the rat CB under physiological conditions, and their expression is down-regulated by losartan treatment.
Effects of angiotensin II on leptin and downstream leptin signaling in the carotid body during acute intermittent hypoxia
2015, Neuroscience
Citation Excerpt :
The carotid body has been shown not only to contain ANG II binding sites (Allen, 1998), but to also possess its own intrinsic renin–angiotensin system (RAS) (Lam and Leung, 2002), suggesting that ANG II within the carotid bodies may act in an autocrine/paracrine manner. Key elements of the RAS, including protein and mRNA of angiotensinogen as well as mRNA of angiotensin I converting enzyme (ACE) have been localized to the type-I glomus cells (Lam and Leung, 2002). A role for ANG II in chemosensitivity is supported by the finding that gene expression for the ANG II type 1 receptor (AT1R) is up-regulated in the carotid bodies during chronic hypoxia (Leung et al., 2000; Fung et al., 2002), and this increased expression of the AT1R is associated with the increased sensitivity of carotid chemoreceptors (Leung et al., 2000; Fung et al., 2001, 2002).
Angiotensin II (ANG II) is known to promote leptin production and secretion. Although ANG II type 1 receptors (AT₁Rs) and leptin are expressed within the carotid body, it is not known whether AT₁R and leptin are co-expressed in the same glomus cells nor if these peptides are affected within the carotid body by intermittent hypoxia (IH). This study was done to investigate whether ANG II modulated leptin signaling in the carotid body during IH. Rats were treated with captopril (Capt) or the AT₁R blocker losartan (Los) in the drinking water for 3 days prior to being exposed to IH (8h) or normoxia (8h). IH induced increases in plasma ANG II and leptin compared to normoxic controls. Capt treatment abolished the plasma leptin changes to IH, whereas Los treatment had no effect on the IH induced increase in plasma leptin. Additionally, carotid body glomus cells containing both leptin and the long form of the leptin receptor (OB-Rb) were found to co-express AT₁R protein, and IH increased the expression of only AT₁R protein within the carotid body in both Capt- and non-Capt-treated animals. On the other hand, Los treatment did not modify AT₁R protein expression to IH. Additionally, Capt and Los treatment eliminated the elevated carotid body leptin protein expression, and the changes in phosphorylated signal transducer and activator of transcription three protein, the short form of the leptin receptor (OB-R₁₀₀), suppressor of cytokine signaling 3, and phosphorylated extracellular-signal-regulated kinase 1/2 protein expression induced by IH. However, Capt elevated the expression of OB-Rb protein, whereas Los abolished the changes in OB-Rb protein to IH. These findings, taken together with the previous observation that ANG II modifies carotid body chemosensitivity, suggest that the increased circulating levels of ANG II and leptin induced by IH act at the carotid body to alter leptin signaling within the carotid body which in turn may influence chemoreceptor function.
Expressions of angiotensin and cytokine receptors in the paracrine signaling of the carotid body in hypoxia and sleep apnea
2015, Respiratory Physiology and Neurobiology
Arterial chemoreceptors in the carotid body are central to the chemical control of breathing in the chemotransduction of physiological stimuli in the arterial blood for eliciting the chemoreflex, which mediates the respiratory, cardiovascular and autonomic responses to hypoxia, hypercapnia and acidosis. Recent evidence suggests that signaling molecules locally produced in the carotid body, including angiotensin II and pro-inflammatory cytokines play an important role in the modulation of the activity of carotid chemoreceptors, via the angiotensin and cytokine receptors expressed in the chemosensitive cells in an autocrine–paracrine manner. The carotid chemoreceptor activity is augmented in subjects at high altitude and in patients with sleep-disordered breathing. Maladaptive responses of the paracrine signaling to hypoxia in the carotid body have been proposed to play a pathogenic role in sleep apnea. Specifically, recent findings show significant increases in expressions of angiotensin receptors and components of a local angiotensin-generating system in the carotid body in sustained or intermittent hypoxia, which augments the chemoreceptor activity and also mediates the inflammatory response of the carotid body to hypoxia. In addition, inflammation of the carotid body involves an increased local expression of cytokine receptors and pro-inflammatory cytokines in sustained or intermittent hypoxia. This review aims to summarize the evidence supporting that the upregulated expression of the angiotensin receptors and cytokine pathways in the carotid body leads to augmented activities of the carotid chemoreceptor in hypoxic conditions, which could play a role in the pathophysiology of sleep apnea.
Histochemical demonstration of tripeptidyl aminopeptidase I in the rat carotid body
2015, Acta Histochemica
Citation Excerpt :
It has been proposed that upon exposure to hypoxia or acid hypercapnia the chemoreceptor glomus cells may release a variety of neurotransmitters, including catecholamines and neuropeptides, which contribute to the increased sympathetic outflow to systemic vascular beds and to a heightened action potential activity on the carotid sinus nerve (Nurse, 2005; Nurse and Piskuric, 2013). It has been revealed that a locally generated Ang system exists in the CB under physiological conditions and that glomus cells express both Ang and its receptors, AT1R and AT2R (Allen, 1998; Lam and Leung, 2002). There is current evidence that multiple intermediate metabolites of this system and their corresponding receptors can influence the glomus cell function and afferent chemoreceptor activity (reviewed in Schultz, 2011).
Tripeptidyl aminopeptidase I (TPP I) is a lysosomal exopeptidase that is widely distributed throughout the central nervous system (CNS) and internal organs in many mammalian species. The enzyme is involved in the breakdown of collagen and different peptides. The carotid body (CB) is the main peripheral arterial chemoreceptor playing an important role in the control of breathing and the autonomic control of cardiovascular function. In response to hypoxia its neuron-like glomus cells release a variety of peptide transmitters that trigger an action potential through the afferent fibers, thus conveying the chemosensory information to the CNS. In the present study we investigated the histochemical localization of TPP I in the CB of rats. Enzyme histochemistry showed high activity of TPP I in CB glomeruli. In particular, the glomus cells contained many TPP I-positive granules, while the glial-like sustentacular cells displayed a slightly fainter reaction. The interglomerular connective tissue was also weakly stained. The results show that both the parenchymal cells of the rat CB express, albeit with different intensity, TPP I. Taken together with our previous enzyme histochemical investigations on the rat CB, it seems likely that the glomus cells possess enzymatic equipment necessary for the neuropeptide intracellular and collagen extracellular initial degradation. These findings also suggest that TPP I is involved in the general turnover of chemotransmitters between glomus cells and sensory nerve endings which emphasizes its importance for chemoreception under hypoxic conditions.

View all citing articles on Scopus

View full text

A locally generated angiotensin system in rat carotid body

Abstract

Introduction

Section snippets

Experimental animals and isolation of carotid body

Expression and localization of angiotensinogen mRNA

Discussion

Acknowledgements

Mol. Cell. Endocrinol.

Regul. Pept.

Anal. Biochem.

Neuroscience

Life Sci.

Biochim. Biophys. Acta

Carotid body: structure and function

Physiol. Rev.

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Am. J. Physiol.

Role of potassium channels in hypoxic chemoreception in rat carotid body type-I cells

Adv. Exp. Med. Biol.

Possible role of coupling between glomus cells in carotid body chemoreception

Biol. Signals

Hypoxia decreases intracellular calcium in adult rat carotid body cells

J. Neurophysiol.

Developmental aspects of oxygen sensing by the carotid body

J. Appl. Physiol.

Cellular mechanisms involved in carotid body inhibition produced by atrial natriuretic peptide

Am. J. Physiol.

Carotid body chemoreceptors: from natural stimuli to sensory discharges

Physiol. Rev.

Peripheral chemoreceptors and cardiovascular regulation

Physiol. Rev.

Renin–angiotensin system: biochemistry and mechanisms of action

Physiol. Rev.

Morphology, physiology, and molecular biology of renin secretion

Physiol. Rev.

The angiotensin II receptors

Pharmacol. Rev.