Endogenous hydrogen sulfide maintains eupnea in an in situ arterially perfused preparation of rats.

Hydrogen sulfide (H2S) is constitutively generated in the human body and works as a gasotransmitter in synaptic transmission. In this study, we aimed to evaluate the roles of endogenous H2S in generating eupnea at the respiratory center. We employed an in situ arterially perfused preparation of decerebrated rats and recorded the central respiratory outputs. When the H2S-producing enzyme cystathionine ß-synthase (CBS) was inhibited, respiration switched from the 3-phase eupneic pattern, which consists of inspiration, postinspiration, and expiration, to gasping-like respiration, which consists of inspiration only. On the other hand, when H2S synthesis was inhibited via cystathionine γ-lyase (CSE) or when H2S synthesis was activated via CBS, eupnea remained unchanged. These results suggest that H2S produced by CBS has crucial roles in maintaining the neuronal network to generate eupnea. The mechanism of respiratory pattern generation might be switched from a network-based system to a pacemaker cell-based system in low H2S conditions.

Irradiation of carotid baroreceptor with low-intensity pulsed ultrasound exerts different metabolic protection in perirenal, epididymal white adipose tissue and interscapular brown adipose tissue of obese rats.

This study was designed to clarify whether the irradiation of carotid baroreceptor (CB) with low-intensity pulsed ultrasound (LIPUS) protects against obesity by rebalancing the autonomic nervous system (ANS). Obesity was induced using a high-fat diet (HFD) for 8 weeks in Sprague-Dawley rats. Irradiation with LIPUS was daily (20 minutes a day) applied to the right CB. In our study, LIPUS significantly ameliorated metabolic disorders in obese rats. LIPUS partly restored norepinephrine (NE) and acetylcholine (ACH) levels in the perirenal white adipose tissue (PWAT), epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT), and plasma of obese rats. LIPUS partially rectified the dysregulated AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor (PPAR) α/É£ pathway in the PWAT, EWAT, and IBAT of obese rats. PPARγ and PPARγ target genes respond more sensitively to HFD and LIPUS in PWAT and EWAT than in IBAT. NE, ACH, uncoupling protein-1, phosphorylated AMPK, PPARα, and PPARα target genes respond more sensitively to HFD and LIPUS in IBAT than in PWAT and EWAT. Conclusion: LIPUS irradiation of CB exerts different metabolic protection in PWAT, EWAT, and IBAT by rebalancing the ANS and rectifying the AMPK/PPARα/É£ pathway in obese rats.

Endogenous Hydrogen Sulfide Enhances Carotid Sinus Baroreceptor Sensitivity by Activating the Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1) Channel.

BACKGROUND: We aimed to investigate the regulatory effects of hydrogen sulfide (H2S) on carotid sinus baroreceptor sensitivity and its mechanisms. METHODS AND RESULTS: Male Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) were used in the experiment and were given an H2S donor or a cystathionine-ß-synthase inhibitor, hydroxylamine, for 8 weeks. Systolic blood pressure and the cystathionine-ß-synthase/H2S pathway in carotid sinus were detected. Carotid sinus baroreceptor sensitivity and the functional curve of the carotid baroreceptor were analyzed using the isolated carotid sinus perfusion technique. Effects of H2S on transient receptor potential cation channel subfamily V member 1 (TRPV1) expression and S-sulfhydration were detected. In SHRs, systolic blood pressure was markedly increased, but the cystathionine-ß-synthase/H2S pathway in the carotid sinus was downregulated in comparison to that of Wistar-Kyoto rats. Carotid sinus baroreceptor sensitivity in SHRs was reduced, demonstrated by the right and upward shift of the functional curve of the carotid baroreceptor. Meanwhile, the downregulation of TRPV1 protein was demonstrated in the carotid sinus; however, H2S reduced systolic blood pressure but enhanced carotid sinus baroreceptor sensitivity in SHRs, along with TRPV1 upregulation in the carotid sinus. In contrast, hydroxylamine significantly increased the systolic blood pressure of Wistar-Kyoto rats, along with decreased carotid sinus baroreceptor sensitivity and reduced TRPV1 protein expression in the carotid sinus. Furthermore, H2S-induced enhancement of carotid sinus baroreceptor sensitivity of SHRs could be amplified by capsaicin but reduced by capsazepine. Moreover, H2S facilitated S-sulfhydration of TRPV1 protein in the carotid sinus of SHRs and Wistar-Kyoto rats. CONCLUSIONS: H2S regulated blood pressure via an increase in TRPV1 protein expression and its activity to enhance carotid sinus baroreceptor sensitivity.

Oxidative stress augments chemoreflex sensitivity in rats exposed to chronic intermittent hypoxia.

Chronic exposure to intermittent hypoxia (CIH) elicits plasticity of the carotid sinus and phrenic nerves via reactive oxygen species (ROS). To determine whether CIH-induced alterations in ventilation, metabolism, and heart rate are also dependent on ROS, we measured responses to acute hypoxia in conscious rats after 14 and 21 d of either CIH or normoxia (NORM), with or without concomitant administration of allopurinol (xanthine oxidase inhibitor), combined allopurinol plus losartan (angiotensin II type 1 receptor antagonist), or apocynin (NADPH oxidase inhibitor). Carotid body nitrotyrosine production was measured by immunohistochemistry. CIH produced an increase in the ventilatory response to acute hypoxia that was virtually eliminated by all three pharmacologic interventions. CIH caused a robust increase in carotid body nitrotyrosine production that was greatly attenuated by allopurinol plus losartan and by apocynin but unaffected by allopurinol. CIH caused a decrease in metabolic rate and a reduction in hypoxic bradycardia. Both of these effects were prevented by allopurinol, allopurinol plus losartan, and apocynin.

Coumestrol inhibits carotid sinus baroreceptor activity by cAMP/PKA dependent nitric oxide release in anesthetized male rats.

Phytoestrogens could offer multiple beneficial effects on the cardiovascular system. Here, we have examined the effects of coumestrol (CMT) on carotid baroreceptors activity (CBA) and the possible mechanisms in male rats. The functional parameters of carotid baroreceptors were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. The levels of protein expression were determined by using ELISA and Western blotting. CMT (1 to 100µmolL(-1)) inhibited CBA, which shifted the functional curve of the carotid baroreceptor to the right and downward, with a marked decrease in the peak slope and the peak integral value of carotid sinus nerve discharge in a concentration dependent manner. These effects were not blocked by a specific estrogen receptor antagonist ICI 182,780, but were completely abolished by nitric oxide (NO) synthase inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester). Furthermore, a NO donor, SIN-1(3-morpholion-sydnon-imine), could potentiate these inhibitory effects of CMT. CMT stimulated the phosphorylation of Ser(1176)-eNOS (endothelial nitric oxide synthase) in a dose-dependent manner in carotid bifurcation tissue over a perfusion period of 15min. The rapid activation of eNOS by CMT was blocked by a highly selective PKA (protein kinase A) inhibitor H89. In addition, inhibition of PI3K (phosphatidylinositol-3-kinase) and ERK (extracellular signal-regulated kinase) pathways had no effect on eNOS activation by CMT. CMT inhibited CBA via eNOS activation and NO synthesis. These effects were mediated by the cAMP/PKA pathway and were unrelated to the estrogenic effect.

Chronic caffeine intake in adult rat inhibits carotid body sensitization produced by chronic sustained hypoxia but maintains intact chemoreflex output.

Sustained hypoxia produces a carotid body (CB) sensitization, known as acclimatization, which leads to an increase in carotid sinus nerve (CSN) activity and ensuing hyperventilation greater than expected from the prevailing partial pressure of oxygen. Whether sustained hypoxia is physiological (high altitude) or pathological (lung disease), acclimatization has a homeostatic implication because it tends to minimize hypoxia. Caffeine, the most commonly ingested psychoactive drug and a nonselective adenosine receptor antagonist, alters CB function and ventilatory responses when administered acutely. Our aim was to investigate the effect of chronic caffeine intake on CB function and acclimatization using four groups of rats: normoxic, caffeine-treated normoxic, chronically hypoxic (12% O2, 15 days), and caffeine-treated chronically hypoxic rats. Caffeine was administered in drinking water (1 mg/ml). Caffeine ameliorated ventilatory responses to acute hypoxia in normoxic animals without altering the output of the CB (CSN neural activity). Caffeine-treated chronically hypoxic rats exhibited a decrease in the CSN response to acute hypoxia tests but maintained ventilation compared with chronically hypoxic animals. The findings related to CSN neural activity combined with the ventilatory responses indicate that caffeine alters central integration of the CB input to increase the gain of the chemoreflex and that caffeine abolishes CB acclimatization. The putative mechanisms involved in sensitization and its loss were investigated: expression of adenosine receptors in CB (A(2B)) was down-regulated and that in petrosal ganglion (A(2A)) was up-regulated in caffeine-treated chronically hypoxic rats; both adenosine and dopamine release from CB chemoreceptor cells was increased in chronic hypoxia and in caffeine-treated chronic hypoxia groups.

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity.

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.

Angiotensin II evokes sensory long-term facilitation of the carotid body via NADPH oxidase.

We previously reported that reactive oxygen species generated by NADPH oxidase 2 (Nox2) induces sensory plasticity of the carotid body, manifested as a progressive increase in baseline sensory activity or sensory long-term facilitation (sLTF). ANG II, a peptide generated within the carotid body, is a potent activator of Nox2. In the present study, we tested the hypothesis that ANG II evokes sLTF of the carotid body via Nox2 activation. Experiments were performed on carotid bodies ex vivo from adult rats and mice. Sensory activity was recorded from the carotid sinus nerve. Repetitive (5 times for 30 s each at 5-min intervals), but not continuous (for 150 s), application of 60 pM ANG II evoked robust sLTF of the carotid body. ACh, ATP, substance P, and KCl, when applied repetitively, stimulated the carotid body but did not evoke sLTF. Reactive oxygen species levels increased in response to repetitive applications of ANG II, and this effect was blocked by apocynin, an inhibitor of Nox2, as well as losartan, an angiotensin type 1 (AT(1)) receptor antagonist. Losartan, apocynin, and 4-(2-aminoethyl)benzenesulfonyl fluoride prevented ANG II-induced sLTF, which was absent in mice deficient in gp91(phox), the catalytic subunit of the Nox2 complex. These results demonstrate that repetitive application of ANG II induces sLTF of the carotid body via activation of Nox2 by AT(1) receptors.

Reflexes from pulmonary arterial baroreceptors in dogs: interaction with carotid sinus baroreceptors.

In contrast to the reflex vasodilatation occurring in response to stimulation of baroreceptors in the aortic arch, carotid sinuses and coronary arteries, stimulation of receptors in the wall of pulmonary arteries results in reflex systemic vasoconstriction. It is rare for interventions to activate only one reflexogenic region, therefore we investigated how these two types of reflexes interact. In anaesthetized dogs connected to cardiopulmonary bypass, reflexogenic areas of the carotid sinuses, aortic arch and coronary arteries and the pulmonary artery were subjected to independently controlled pressures. Systemic perfusion pressure (SPP) measured in the descending aorta (constant flow) provided an index of systemic vascular resistance. In other experiments, sympathetic efferent neural activity was recorded in fibres dissected from the renal nerve (RSNA). Physiological increases in pulmonary arterial pressure (PAP) induced significant increases in SPP (+39.1 ± 10.4 mmHg) and RSNA (+17.6 ± 2.2 impulses s(−1)) whereas increases in carotid sinus pressure (CSP) induced significant decreases in SPP (−42.6 ± 10.8 mmHg) and RSNA (−42.8 ± 18.2 impulses s(−1)) (P < 0.05 for each comparison; paired t test). To examine possible interactions, PAP was changed at different levels of CSP in both studies. With CSP controlled at 124 ± 2 mmHg, the threshold, 'set point' and saturation pressures of the PAP­SPP relationship were higher than those with CSP at 60 ± 1 mmHg; this rightward shift was associated with a significant decrease in the reflex gain. Similarly, increasing CSP produced a rightward shift of the PAP­RSNA relationship, although the effect on reflex gain was inconsistent. Furthermore, the responses to changes in CSP were influenced by setting PAP at different levels; increasing the level of PAP from 5 ± 1 to 33 ± 3 mmHg significantly increased the set point and threshold pressures of the CSP­SPP relationship; the reflex gain was not affected. These results indicate the existence of interaction between pulmonary arterial and carotid sinus baroreceptor reflexes; physiological and pathological states that alter the stimulus to one may alter the reflex responses from the other.

Catecholamine release from isolated sensory neurons of cat petrosal ganglia in tissue culture.

The petrosal ganglion (PG) is entirely constituted by the perikarya of primary sensory neurons, part of which innervates the carotid body via the carotid sinus nerve (CSN). Application of acetylcholine (ACh) or nicotine (Nic) as well as adenosine 5'-triphosphate (ATP) to the PG in vitro increases the frequency of CSN discharges, an effect that is modified by the concomitant application of dopamine (DA). Since a population of PG neurons expresses tyrosine hydroxylase, and DA is released from the cat carotid body in response to electrical stimulation of C-fibers in the CSN, it is possible that DA may be released from the perikarya of PG neurons. Therefore, we studied whether ACh or Nic, ATP and high KCl could induce DA release from PG neurons in culture. Petrosal ganglia were excised from pentobarbitone-anesthetized adult cats, dissociated and their neurons maintained in culture for 7-21 days. Catecholamine release was measured by amperometry via carbon-fiber microelectrodes. In response to KCl, Nic, ACh or ATP application, about 25% of neurons exhibited electrochemical signals compatible with DA release. This percentage increased to 41% after loading the neurons with exogenous DA. The present results suggest that DA release may be induced from the perikarya of a population of PG neurons.

Effects of selective sinoaortic denervations on phenylephrine-induced activational responses in the nucleus of the solitary tract.

Intravenous administration of phenylephrine provokes a pattern of cellular activation in the nucleus of the solitary tract that resembles the central distributions of primary baroreceptor afferents supplied by the carotid sinus and aortic depressor nerves. Transganglionic transport and denervation methods were used in an experimental setting to test the dependence of phenylephrine-induced Fos immunoreactivity on the integrity of buffer nerve afferents, and to identify the subregions of the nucleus of the solitary tract supplied by each. Cholera toxin B-horseradish peroxidase injections into either or both nerves revealed terminal labeling concentrated in, but not restricted to, the dorsal commissural part of the nucleus of the solitary tract at the level of the apex of calamus scriptorius, and extending into the dorsal subnucleus at the level of the area postrema. Preferential ramifications of carotid sinus and aortic depressor nerve afferents at the levels of the commissural part of the nucleus and the area postrema, respectively, were reflected in the extent to which labeled fibers comingled with neurons exhibiting phenylephrine-induced Fos in dual labeling experiments. Complete sinoaortic denervation reduced by 90% the number of neurons exhibiting drug-induced Fos expression. Selective carotid and aortic sinus denervations effected partial reductions manifest preferentially in the caudal and rostral foci of the distribution, respectively. Reduced activational responses at the level of the area postrema of aortic sinus-denervated rats were accompanied by a reduction in cellular nicotinamide adenine dinucleotide phosphate-diaphorase activity in this region. Animals killed 30 days after complete sinoaortic denervation displayed no evidence of recovery of phenylephrine-induced Fos, while the strength and distribution of the response in rats that received selective carotid sinus denervation were indistinguishable from those seen in controls. These findings (i) support the dependence of phenylephrine-induced Fos expression on the integrity of carotid sinus and aortic depressor nerve afferents, (ii) provide anatomical and functional evidence that the two buffer nerves distribute differentially within the nucleus of the solitary tract, and (iii) implicate central reorganization as a likely basis for functional recovery of baroreflex mechanisms following partial sinoaortic denervation.

Thromboxane A2 does not act at the carotid sinus to mediate cardiovascular, adrenocorticotropin, cortisol, or blood gas responses.

Thromboxane A2 (TxA2), well known as a vasoconstrictor and activator of platelets, also stimulates reflex cardiovascular, pituitary, adrenocortical, and blood gas responses, although the site of action is unknown. Previously we determined that the site of these actions is perfused by the carotid vasculature. The purpose of the present study was to test the hypothesis that TxA2 stimulates these responses by acting at the carotid sinus. The TxA2 mimetic U46619 (1 microgram.kg-1.min-1) or saline was infused into the carotid artery (CA) or vena cava in conscious, chronically instrumented carotid sinus denervated (CSD) or sham-operated sheep. Mean arterial pressure increased in all groups receiving U46619. Heart rate increased only in the CSD group receiving CA infusions of U46619. Adrenocorticotropic hormone (ACTH) and cortisol increased in the sham and CSD groups receiving CA U46619, and responses were not different between sham and CSD groups. PaCO2 values were higher in all CSD treatment groups compared with sham treatment groups. Arterial pH increased and PaCO2 decreased in both the sham and CSD groups in response to CA U46619. Although PaCO2 values were higher overall in the CSD group, the magnitude of change in response to U46619 infusions was similar in sham and CSD animals. There was no difference in pHa between CSD and sham groups. Hematocrit and PaO2 did not change. We conclude that TxA2 does not act at the carotid sinus, as responses to U46619 infusions in CSD animals were not different in the cases of ACTH, cortisol, and blood gases, or were enhanced rather than diminished in the case of heart rate. These findings support a hypothesis that TxA2 acts at the brain to mediate cardiovascular, pituitary, adrenocortical, and blood gas responses.

Gene transfer to carotid sinus in vivo: a novel approach to investigation of baroreceptors.

Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch. The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia. Replication-deficient adenovirus containing the gene for Escherichia coli beta-galactosidase (beta-Gal) was applied topically to the carotid sinuses of anesthetized rabbits. Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light). Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg. Beta-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days. Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses. Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses. In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation. The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.

Precise coexistence of regulatory peptides in the nerve fibers of the amphibian carotid labyrinth demonstrated by a combination of double immunofluorescence labelling and a multiple dye filter.

An application of double-immunolabelling in combination with a multiple dye filter system demonstrated new findings regarding the distribution pattern of peptidergic fibers in the carotid labyrinth to addition to our previous findings shown by the individual filter system. In high magnification images of about 10% of the yellowish fibers which represent the coexistence of two neuropeptides, there was a definite difference in localization between the fluorescence originating from rhodamine (substance P fibers) and from FITC (vasoactive intestinal polypeptide and neuropeptide Y fibers), but it was clear that they are intertwined within a single nerve bundle. This combination method was able to discriminate two different peptidergic fibers which run side by side. The coexistence suggested previously by the individual filter system may actually be due to the phenomenon described above. This means that it is necessary to apply the multiple dye filter system for reliable evidence of coexistence of different two substances in a single nerve fiber.

Peptidergic innervation in the amphibian carotid labyrinth.

The amphibian carotid labyrinth, which corresponds to the mammalian carotid body and carotid sinus, is innervated by nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and somatostatin (SOM). SP, CGRP, VIP, and NPY immunoreactive varicose fibers are more densely distributed in the peripheral portion of the carotid labyrinth than FMRF and SOM fibers. The time of appearance of SP, CGRP, and VIP is different for each. First CGRP fibers, then SP fibers appear at an early stage of larval development, and finally VIP fibres are detected at a later stage of larval development. Most SP fibres show coexistence with CGRP, and some SP fibres which show coexistence with NPY immunoreactivity are assumed to be continuous with those demonstrating VIP immunoreactivity. This indicates the possibility of coexistence of four different peptides in the same nerve fibers within the labyrinth. In various vasculatures of mammals, it has been shown that SP, CGRP, VIP, and NPY have a vasoactive nature in relation to the vascular smooth muscle cells. On this basis, it seems that the target of the peptidergic innervation in the amphibian carotid labyrinth is the smooth muscle cells which are abundantly distributed in the intervascular stroma. Accordingly, the peptidergic innervation may be involved in the vascular regulatory function of the labyrinth, although the possibility that these peptides participate in the chemoreception cannot be ruled out. In addition, the vascular regulatory function of the labyrinth may be modulated by the interaction of multiple neuropeptides.