Role of endothelin and endothelin A-type receptor in adaptation of the carotid body to chronic hypoxia

Chronic exposure in a low-PO(2) environment (i.e., chronic hypoxia, CH) elicits an elevated hypoxic ventilatory response and increased hypoxic chemosensitivity in arterial chemoreceptors in the carotid body. In the present study, we examine the hypothesis that changes in chemosensitivity are mediated by endothelin (ET), a 21-amino-acid peptide, and ET(A) receptors, both of which are normally expressed by O(2)-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ET(A) expression in type I cells after 3, 7, and 14 days of CH (380 Torr). Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked by acute hypoxia. In normal carotid body, the specific ET(A) antagonist BQ-123 (5 microM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the hypoxia-evoked discharge by 20% (P < 0.01). This inhibitory effect progressed to 45% at day 9 of CH and to nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the presence of BQ-123, the magnitude of the activity evoked by hypoxia did not differ in normal vs. CH preparations, indicating that the increased activity was the result of endogenous ET acting on an increasing number of ET(A). Collectively, our data suggest that ET and ET(A) autoreceptors on O(2)-sensitive type I cells play a critical role in CH-induced increased chemosensitivity in the rat carotid body.     

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice

Introduction  

Endothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice.     

Effect of endothelin dual receptor antagonist on VEGF levels in streptozotocin-induced diabetic rat retina

Diabetic retinopathy (DR), one of the most serious causes of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in retina. Recently, leukocyte adhesion (leukostasis) is blamed for the occlusion of retinal capillary vascularity, which ultimately contributes to the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in the diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is deeply linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study yet has reported whether ET-1 dual receptor antagonist could alter the upregulated VEGF and ICAM-1 levels in the diabetic retina. The present study investigated the effect of ET(A/B) dual receptor antagonist (SB209670; 1 mg/rat/day) on the expression of VEGF and ICAM-1 in the diabetic rat retina. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 65 mg/kg) in Sprague-Dawley rats, whereas control rats (non-DM control) received only citrate buffer. After 1 week, the STZ-administered rats were randomly divided into two groups: one group (DM+SB209670) received ET(A/B) dual receptor antagonist for 2 weeks, and a vehicle group (DM+vehicle) was treated only with saline. After the treatment period, the retinas were removed from the eyeballs. In DM+vehicle group, the VEGF expression of the retinas was significantly increased (32.8 pg/mg) in comparison to that in the non-DM control group (26.2 pg/mg); this upregulation of VEGF was reversed in the DM+SB209670 group (28.6 pg/mg). The expression of retinal ICAM-1 was increased in the DM+vehicle group (152.2 pg/mg) compared with the non-DM control group (121.6 pg/mg). However, SB209670 treatment did not alter the expression of retinal ICAM-1 level (154.8 pg/ml) in DM rats. Thus we conclude that an ET(A/B) dual receptor antagonist could reverse the expression level of VEGF in the diabetic retina while failing to normalize the upregulated ICAM-1 expression.     

Effect of chronic hypoxia on cholinergic chemotransmission in rat carotid body.

Current views suggest that oxygen sensing in the carotid body occurs in chemosensory type I cells, which excite synaptically apposed chemoafferent nerve terminals in the carotid sinus nerve (CSN). Prolonged exposure in a low-oxygen environment [i.e., chronic hypoxia (CH)] elicits an elevated stimulus-evoked discharge in chemoreceptor CSN fibers (i.e., increased chemosensitivity). In the present study, we evaluated cholinergic chemotransmission in the rat carotid body in an effort to test the hypothesis that CH enhances ACh-mediated synaptic activity between type I cells and chemoafferent nerve terminals. Animals were exposed in a hypobaric chamber (barometric pressure = 380 Torr) for 9-22 days before evaluation of chemoreceptor activity using an in vitro carotid body/CSN preparation. Nerve activity evoked by ACh was significantly larger (P < 0.01) after CH, suggesting increased expression of cholinergic receptors. Approximately 80% of the CSN impulse activity elicited by ACh (100- or 1,000-microg bolus) in both normal and CH preparations was blocked by the specific nicotinic receptor antagonist mecamylamine (100 microM). CSN activity elicited by acute hypoxia or hypercapnia in normal preparations was likewise blocked (> or =80%) in the presence of 100 muM mecamylamine, but after CH the enhanced CSN activity elicited by acute hypoxia or hypercapnia was not reduced in the presence of 100 or 500 microM mecamylamine. A muscarinic receptor antagonist, atropine (10 microM), and a specific nicotinic receptor alpha7 subunit antagonist, methyllycaconatine (50 nM), blocked approximately 50% of the hypoxia-evoked activity in normal preparations but were ineffective after CH. Prolonged exposure to hypoxia appears to dramatically alter chemotransmission in the carotid body, and may induce alternative neurotransmitter mechanisms and/or electrical coupling between type I cells and chemoafferent nerve terminals.     

Effect of chronic hypoxia on purinergic synaptic transmission in rat carotid body.

Recent studies indicate that chemoafferent nerve fiber excitation in the rat carotid body is mediated by acetylcholine and ATP, acting at nicotinic cholinergic receptors and P2X2 purinoceptors, respectively. We previously demonstrated that, after a 10- to 14-day exposure to chronic hypoxia (CH), the nicotinic cholinergic receptor blocker mecamylamine no longer inhibits rat carotid sinus nerve (CSN) activity evoked by an acute hypoxic challenge. The present experiments examined the effects of CH (9-16 days at 380 Torr) on the expression of P2X2 purinoceptors in carotid body and chemoafferent neurons, as well as the effectiveness of P2X2 receptor blocking drugs on CSN activity evoked by hypoxia. In the normal carotid body, immunocytochemical studies demonstrated a dense plexus of P2X2-positive nerve fibers penetrating lobules of type I cells. In addition, type I cells were lightly stained, indicating P2X2 receptor expression. After CH, the intensity of P2X2 receptor immunostaining was maintained in chemosensory type I cells and in the soma of chemoafferent neurons. P2 receptor expression on type I cells was confirmed by demonstrations of ATP-evoked increased intracellular Ca2+; this response was modulated by simultaneous exposure to hypoxia. In normal preparations, CSN activity evoked by hypoxia in vitro was 65% inhibited in the presence of specific P2X2 receptor antagonists. However, unlike the absence of mecamylamine action after CH, P2X2 antagonists remained effective against hypoxia-evoked activity after CH. Our findings indicate that ATP acting at P2X2 receptors contributes to adjusted chemoreceptor activity after CH, indicating a possible role for purinergic mechanisms in the adaptation of the carotid body in a chronic low-O2 environment.     

精氨酸加压素V1受体阻断剂对毒死蜱降温效应的阻断作用

精氨酸加压素 (ＡＶＰ)能够降低致热原性和非致热原性的发热效应 ,精氨酸加压素Ｖ1受体阻断剂 (ＡＶＰＶ1受体阻断剂 )可以阻断其作用。最近几年的研究发现 ,有机磷农药杀虫剂毒死蜱 (ＣＨＰ)可以引起体温降低。目前认为ＣＨＰ引起的降温效应与体内乙酰胆硷 (Ａｃｈ)含量增加有关 ,因为Ａｃｈ参与体温调节过程。我们考虑既然ＡＶＰ有参与体温调节和降温过程 ,那么ＣＨＰ的降温作用是否与ＡＶＰ有关 ,所以本研究观察了ＡＶＰＶ1受体阻断剂对ＣＨＰ引起的体温变化的影响。1　材料与方法(1)动物与试剂　实验用 2月龄的ＳＤ雄性和雌性大鼠 ,体重…     

Ultrastructural changes in the rat carotid body in severe haemorrhagia

Summary Rat carotid bodies were studied electron microscopically after short-term severe hypovolaemia, which is known to induce a marked chemoreceptor activation in the carotid body. Altogether 84 nerve-endings in the hypovolaemic rats' carotid bodies and 91 nerve-endings in the control carotid bodies were investigated. An increased accumulation of the glomus cell granular vesicles near the synaptic specializations of the nerve-endings was observed after hypovolaemia. Moreover, a statistically significant increase in the contacts between the nerve-ending synaptic specializations and the glomus cell granular vesicles was observed after hypovolaemia. A suggestion was made that the glomus cells might act as modulating, probably inhibitory, interneurones, whose catecholamines are responsible for the inhibition.The authors are greatly indebted to lecturer Pekka Korkala Ph.L. from the Department of Psychology for his skilful statistical analysis of the results.     

Endothelin A and B receptor antagonist bosentan reduces postischemic myocardial injury in the rat: critical timing of administration

The purpose of this study was to investigate the effects of bosentan, a mixed endothelin receptor A and B subtype antagonist, on myocardial ischemia-reperfusion injury and to explore the influence of the timing of bosentan administration on its cardioprotective effects. Adult rat hearts were perfused by the Langendorff technique with Krebs-Henseleit solution (KH) at a constant flow rate at 10 mL/min. Global myocardial ischemia was induced by stopping KH perfusion for 40 min, and this was followed by 60 min of reperfusion. Hearts were randomized to 1 of 3 experimental groups (n = 7 each): untreated control; treatment with bosentan 1 micromol/L 10 min prior to, during 40 min global ischemia, and for 15 min of reperfusion (BOS); or treatment with bosentan 1 micromol/L after 15 min of reperfusion (BOS-R). We observed that BOS-R, but not the BOS treatment regimen, significantly reduced the release of cardiac-specific creatine kinase and postischemic myocardial infarct size (P < 0.05 vs. control) without affecting myocardial contractility. Left ventricular developed pressure in the BOS group was significantly (P < 0.01) lower than that in the control group throughout reperfusion. It is concluded that pharmacologically delayed antagonism of endothelin-1 during reperfusion attenuates postischemic myocardial injury. Endothelin-1 antagonist application during early reperfusion may exacerbate postischemic myocardial dysfunction.     

Chronic intermittent hypoxia-induced vascular enlargement and VEGF upregulation in the rat carotid body is not prevented by antioxidant treatment

Chronic intermittent hypoxia (CIH), a characteristic of sleep obstructive apnea, enhances carotid body (CB) chemosensory responses to hypoxia, but its consequences on CB vascular area and VEGF expression are unknown. Accordingly, we studied the effect of CIH on CB volume, glomus cell numbers, blood vessel diameter and number, and VEGF immunoreactivity (VEGF-ir) in male Sprague-Dawley rats exposed to 5% O(2), 12 times/h for 8 h or sham condition for 21 days. We found that CIH did not modify the CB volume or the number of glomus cells but increased VEGF-ir and enlarged the vascular area by increasing the size of the blood vessels, whereas the number of the vessels was unchanged. Because oxidative stress plays an essential role in the CIH-induced carotid chemosensory potentiation, we tested whether antioxidant treatment with ascorbic acid may impede the vascular enlargement and the VEGF upregulation. Ascorbic acid, which prevents the CB chemosensory potentiation, failed to impede the vascular enlargement and the increased VEGF-ir. Thus present results suggest that the CB vascular enlargement induced by CIH is a direct effect of intermittent hypoxia and not secondary to the oxidative stress. Accordingly, the subsequent capillary changes may be secondary to the mechanisms involved in the neural chemosensory plasticity induced by intermittent hypoxia.     

Effect of chronic endothelin receptor antagonism on cerebrovascular function in type 2 diabetes

Diabetes increases the risk of stroke and contributes to poor clinical outcomes in this patient population. Myogenic tone of the cerebral vasculature, including basilar arteries, plays a key role in controlling cerebral blood flow. Increased myogenic tone is ameliorated with ET receptor antagonism in Type 1 diabetes. However, the role of endothelin-1 (ET-1) and its receptors in cerebrovascular dysfunction in Type 2 diabetes, a common comorbidity in stroke patients, remains poorly elucidated. Therefore, we hypothesized that 1) cerebrovascular dysfunction occurs in the Goto-Kakizaki (GK) model of Type 2 diabetes, and 2) pharmacological antagonism of ETA receptors ameliorates, while ETB receptor blockade augments vascular dysfunction. GK or control rats were treated with antagonists to either ETA (atrasentan, 5 mg.kg(-1).day(-1)) or ETB (A-192621, 15 or 30 mg.kg(-1).day(-1)) receptors for 4 wk and vascular function of basilar arteries was assessed using a wire myograph. GK rats exhibited increased sensitivity to ET-1. ET(A) receptor antagonism caused a rightward shift, indicating decreased sensitivity in diabetes, while it increased sensitivity to ET-1 in control rats. Endothelium-dependent relaxation was impaired in diabetes. ETA receptor blockade restored relaxation to control values in the GK animals with no significant effect in Wistar rats and ETB blockade with 30 mg.kg(-1).day(-1) A-192621 caused paradoxical constriction in diabetes. These studies demonstrate that cerebrovascular dysfunction occurs and may contribute to altered regulation of myogenic tone and cerebral blood flow in diabetes. While ETA receptors mediate vascular dysfunction, ETB receptors display differential effects. These results underscore the importance of ETA/ETB receptor balance and interactions in cerebrovascular dysfunction in diabetes.     

Sustained hypoxia-induced proliferation of carotid body type I cells in rats.

Sustained hypoxia (SH) has been shown to cause profound morphological and cellular changes in carotid body (CB). However, results regarding whether SH causes CB type I cell proliferation are conflicting. By using bromodeoxyuridine, a uridine analog that is stably incorporated into cells undergoing DNA synthesis, we have found that SH causes the type I cell proliferation in the CB; the proliferation occurs mainly during the first 1-3 days of hypoxic exposure. Moreover, the new cells survive for at least 1 mo after the return to normoxia. Also, SH does not cause any cell death in CB as examined by the terminal deoxynucleotidyl transferase-mediated dUTP-X nick-end labeling assay. Taken together, our results suggest that SH stimulates CB type I cell proliferation, which may produce long-lasting changes in CB morphology and function.     

Effect of chronic and selective endothelin receptor antagonism on microvascular function in type 2 diabetes

Vascular dysfunction, which presents either as an increased response to vasoconstrictors or an impaired relaxation to dilator agents, results in worsened cardiovascular outcomes in diabetes. We have established that the mesenteric circulation in Type 2 diabetes is hyperreactive to the potent vasoconstrictor endothelin-1 (ET-1) and displays increased nitric oxide-dependent vasodilation. The current study examined the individual and/or the relative roles of the ET receptors governing vascular function in the Goto-Kakizaki rat, a mildly hyperglycemic, normotensive, and nonobese model of Type 2 diabetes. Diabetic and control rats received an antagonist to either the ET type A (ETA; atrasentan; 5 mg x kg(-1) x day(-1)) or type B (ET(B); A-192621; 15 or 30 mg x kg(-1) x day(-1)) receptors for 4 wk. Third-order mesenteric arteries were isolated, and vascular function was assessed with a wire myograph. Maximum response to ET-1 was increased in diabetes and attenuated by ETA antagonism. ETB blockade with 15 mg/kg A-192621 augmented vasoconstriction in controls, whereas it had no further effect on ET-1 hyperreactivity in diabetes. The higher dose of A-192621 showed an ETA-like effect and decreased vasoconstriction in diabetes. Maximum relaxation to acetylcholine (ACh) was similar across groups and treatments. ETB antagonism at either dose had no effect on vasorelaxation in control rats, whereas in diabetes the dose-response curve to ACh was shifted to the right, indicating a decreased relaxation at 15 mg/kg A-192621. These results suggest that ETA receptor blockade attenuates vascular dysfunction and that ETB receptor antagonism exhibits differential effects depending on the dose of the antagonists and the disease state.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Strong expression of interleukin-1 receptor type I in the rat carotid body.

One of the unsolved key questions in neuroimmunomodulation is how peripheral immune signals are transmitted to the brain. It has been reported that the vagus might play a role in this regard. The underlying mechanism for this immune system-to-brain communication route is related to the binding of cytokines, such as interleukin (IL)-1beta originating from activated immune cells, to their receptors in glomus cells of the vagal paraganglia. The existence of IL-1 receptor type I (IL-1RI) in vagal paraganglia has been proved. On the basis of these studies, a hypothesis is raised that the carotid body, as the largest paraganglion, might play a similar role to that of its abdominal partner. In this study we examined the distribution of IL-1RI in the carotid body by immunohistochemistry (IHC) and Western blotting techniques. The IHC results showed that almost all glomus cells in the carotid body displayed strong IL-1RI immunoreactivity. The IL-1RI-immunoreactive products were localized in the cytoplasm, nucleus, and cell membrane of the glomus cells. The Western blotting results also confirmed the existence of IL-1RI in both membranous and cytoplasmic elements of the carotid body. These results imply that the carotid body not only serves as a chemoreceptor for modulation of cardiorespiratory performance, as traditionally recognized, but also acts as a cytokine chemorereceptor for sensing immune signals.     

Influence of endothelin 1 receptor inhibition on functional, structural and molecular changes in the rat heart after irradiation

Radiation-induced heart disease is a severe side effect of thoracic radiotherapy. Studies suggest that mast cells play a protective role in radiation-induced heart disease and that the endothelin (ET) system mediates protective effects of mast cells in other disorders. This study examined whether mast cells modulate the cardiac ET system and examined the effects of ET receptor inhibition in a rat model of radiation-induced heart disease. Mast cell-deficient (Ws/Ws), mast cell-competent (+/+) and Sprague-Dawley rats received 18 Gy irradiation to the heart. Left ventricular mRNA of ET1 and its receptors (ETA and ETB) was measured in Ws/Ws and +/+ rats at 1 week and 3 months. Sprague-Dawley rats were treated with the ETA/ETB antagonist bosentan, and at 6 months cardiac changes were assessed using the Langendorff perfused rat heart preparation, immunohistochemistry and real-time PCR. Ws/Ws and +/+ rat hearts did not differ in baseline mRNA. In contrast, +/+ rats hearts exhibited up-regulation of ET1 after irradiation, whereas Ws/Ws rats hearts did not, suggesting the possibility of interactions between mast cells and the cardiac ET system. Bosentan induced reductions in left ventricular systolic pressure, developed pressure and +dP/dtmax but did not affect fibrosis. Because of the known opposing effects of ETA and ETB, studies with selective antagonists may clarify the role of each receptor.     

Hypoxic adaptation of the rat carotid body

Three types of hypoxia with different levels of carbon dioxide (hypocapnic, isocapnic, and hypercapnic hypoxia) have been called systemic hypoxia. The systemic hypoxic carotid bodies were enlarged several fold, but the degree of enlargement was different for each. The mean short and long axes of hypocapnic and isocapnic hypoxic carotid bodies were 1.6 (short axis) and 1.8-1.9 (long axis) times larger than normoxic control carotid bodies, respectively. Those of hypercapnic hypoxic carotid bodies were 1.2 (short axis) and 1.5 (long axis) times larger than controls, respectively. The rate of enlargement in hypercapnic hypoxic carotid bodies was lower than in hypocapnic and isocapnic hypoxic carotid bodies. The rate of vascular enlargement in hypercapnic hypoxic carotid bodies was also smaller than in hypocapnic and isocapnic hypoxic carotid bodies. Thus, the enlargement of hypoxic carotid bodies is mainly due to vascular dilation. Different levels of arterial CO2 tension change the peptidergic innervation during chronically hypoxic exposure. The characteristic vascular arrangement was under the control of altered peptidergic innervation. During the course of hypoxic adaptation, the enlargement of the carotid bodies with vascular expansion began soon after the start of hypoxic exposure. During the course of recovery, the shrinking of the carotid bodies with vascular contraction also started at a relatively early period after the termination of chronic hypoxia. These processes during the course of hypoxic adaptation and during the course of recovery were under the control of peptidergic innervation. These findings may provide a standard for further studies of hypoxic carotid bodies.     

Effect of cycloheximide on the morphological and biochemical changes in chromatin in non-irradiated and irradiated rat thymocytes

The level of chromatin degradation was studied and the method of electron-microscopy was used to estimate the changes in the ultrastructure of irradiated and nonirradiated thymocytes of rats treated with cycloheximide. The latter was found to decrease the degree of derangement of nuclear ultrastructure and the level of chromatin degradation in exposed animals and to increase the yield of these damages in thymocytes of nonirradiated animals. The electronmicroscopic determinations showed that the percentage of thymocytes with the impaired nucleus structure is twice as high as that of degraded chromatin. The causes of the quantitative disagreement between the morphological and biochemical indices of the interphase thymocyte death are discussed.