Peripheral chemoreceptors and cardiorespiratory coupling: a link to sympatho-excitation

Chronic intermittent hypoxia (CIH) has been identified as a relevant risk factor for the development of enhanced sympathetic outflow and arterial hypertension. Several studies have highlighted the importance of peripheral chemoreceptors for the cardiovascular changes elicited by CIH. However, the effects of CIH on the central mechanisms regulating sympathetic outflow are not fully elucidated. Our research group has explored the hypothesis that the enhanced sympathetic drive following CIH exposure is, at least in part, dependent on alterations in the respiratory network and its interaction with the sympathetic nervous system. In this report, I discuss the changes in the discharge profile of baseline sympathetic activity in rats exposed to CIH, their association with the generation of active expiration and the interactions between expiratory and sympathetic neurones after CIH conditioning. Together, these findings are consistent with the theory that mechanisms of central respiratory–sympathetic coupling are a novel factor in the development of neurogenic hypertension.

The nucleus of the solitary tract and the coordination of respiratory and sympathetic activities

It is well known that breathing introduces rhythmical oscillations in the heart rate and arterial pressure levels. Sympathetic oscillations coupled to the respiratory activity have been suggested as an important homeostatic mechanism optimizing tissue perfusion and blood gas uptake/delivery. This respiratory-sympathetic coupling is strengthened in conditions of blood gas challenges (hypoxia and hypercapnia) as a result of the synchronized activation of brainstem respiratory and sympathetic neurons, culminating with the emergence of entrained cardiovascular and respiratory reflex responses. Studies have proposed that the ventrolateral region of the medulla oblongata is a major site of synaptic interaction between respiratory and sympathetic neurons. However, other brainstem regions also play a relevant role in the patterning of respiratory and sympathetic motor outputs. Recent findings suggest that the neurons of the nucleus of the solitary tract (NTS), in the dorsal medulla, are essential for the processing and coordination of respiratory and sympathetic responses to hypoxia. The NTS is the first synaptic station of the cardiorespiratory afferent inputs, including peripheral chemoreceptors, baroreceptors and pulmonary stretch receptors. The synaptic profile of the NTS neurons receiving the excitatory drive from afferent inputs is complex and involves distinct neurotransmitters, including glutamate, ATP and acetylcholine. In the present review we discuss the role of the NTS circuitry in coordinating sympathetic and respiratory reflex responses. We also analyze the neuroplasticity of NTS neurons and their contribution for the development of cardiorespiratory dysfunctions, as observed in neurogenic hypertension, obstructive sleep apnea and metabolic disorders.

Sympatho-excitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Bötzinger complex of rats

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Bötzinger/pre-Bötzinger complexes (RVLM/BötC/pre-BötC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BötC/pre-BötC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BötC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 ± 0.02 vs. 1 ± 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 ± 2 vs. 82 ± 2%; P < 0.05; n = 8), but decreased postinspiratory (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ±1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 ± 6 vs. 40 ± 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 ± 8 vs. 26 ± 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BötC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BötC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BötC and RVLM/pre-BötC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BötC is essential to the coupling of active expiration and sympathoexcitatory response.

Effect of furosemide treatment on the central and peripheral pressor responses to cholinergic and adrenergic agonists, angiotensin II, hypertonic solution and vasopressin

The present study was performed to investigate the effect of treatment with furosemide on the pressor response induced by intracerebroventricular (i.c.v.) injections of cholinergic (carbachol) and adrenergic (norepinephrine) agonists, angiotensin II (ANGII) and hypertonic saline (HS, 2 M NaCl). The changes induced by furosemide treatment on the pressor response to intravenous (i.v.) norepinephrine, ANGII and arginine vasopressin (AVP) were also studied. Rats with a stainless-steel cannula implanted into the lateral ventricle (LV) were used. Two injections of furosemide (30 mg/kg b.wt. each) were performed 12 and 1 h before the experiments. Treatment with furosemide reduced the pressor response induced by carbachol, norepinephrine and ANGII i.c.v., but no change was observed in the pressor response to i.c.v. 2 M NaCl. The pressor response to i.v. ANGII and norepinephrine, but not AVP, was also reduced after treatment with furosemide. These results show that the treatment with furosemide impairs the pressor responses induced by central or peripheral administration of adrenergic agonist or ANGII, as well as those induced by central cholinergic activation. The results suggest that the treatment with furosemide impairs central and peripheral pressor responses mediated by sympathetic activation and ANGII, but not those produced by AVP. © 1992.

Coupling between respiratory and sympathetic activities as a novel mechanism underpinning neurogenic hypertension

Enhanced sympathetic outflow to the heart and resistance vessels greatly contributes to the onset and maintenance of neurogenic hypertension. There is a consensus that the development of hypertension (clinical and experimental) is associated with an impairment of sympathetic reflex control by arterial baroreceptors. More recently, chronic peripheral chemoreflex activation, as observed in obstructive sleep apnea, has been proposed as another important risk factor for hypertension. In this review, we present and discuss recent experimental evidence showing that changes in the respiratory pattern, elicited by chronic intermittent hypoxia, play a key role in increasing sympathetic activity and arterial pressure in rats. This concept parallels results observed in other models of neurogenic hypertension, such as spontaneously hypertensive rats and rats with angiotensin II–salt-induced hypertension, pointing out alterations in the central coupling of respiratory and sympathetic activities as a novel mechanism underlying the development of neurogenic hypertension.

Sympathetic overactivity precedes metabolic dysfunction in a fructose model of glucose intolerance in mice

De Angelis K, Senador DD, Mostarda C, Irigoyen MC, Morris M. Sympathetic overactivity precedes metabolic dysfunction in a fructose model of glucose intolerance in mice. Am J Physiol Regul Integr Comp Physiol 302: R950-R957, 2012. First published February 8, 2012; doi: 10.1152/ajpregu.00450.2011.-Consumption of high levels of fructose in humans and animals leads to metabolic and cardiovascular dysfunction. There are questions as to the role of the autonomic changes in the time course of fructose-induced dysfunction. C57/BL male mice were given tap water or fructose water (100 g/l) to drink for up to 2 mo. Groups were control (C), 15-day fructose (F15), and 60-day fructose (F60). Light-dark patterns of arterial pressure (AP) and heart rate (HR), and their respective variabilities were measured. Plasma glucose, lipids, insulin, leptin, resistin, adiponectin, and glucose tolerance were quantified. Fructose increased systolic AP (SAP) at 15 and 60 days during both light (F15: 123 +/- 2 and F60: 118 +/- 2 mmHg) and dark periods (F15: 136 +/- 4 and F60: 136 +/- 5 mmHg) compared with controls (light: 111 +/- 2 and dark: 117 +/- 2 mmHg). SAP variance (VAR) and the low-frequency component (LF) were increased in F15 (>60% and >80%) and F60 (>170% and >140%) compared with C. Cardiac sympatho-vagal balance was enhanced, while baroreflex function was attenuated in fructose groups. Metabolic parameters were unchanged in F15. However, F60 showed significant increases in plasma glucose (26%), cholesterol (44%), triglycerides (22%), insulin (95%), and leptin (63%), as well as glucose intolerance. LF of SAP was positively correlated with SAP. Plasma leptin was correlated with triglycerides, insulin, and glucose tolerance. Results show that increased sympathetic modulation of vessels and heart preceded metabolic dysfunction in fructose-consuming mice. Data suggest that changes in autonomic modulation may be an initiating mechanism underlying the cluster of symptoms associated with cardiometabolic disease.

Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats

Costa-Silva JH, Zoccal DB, Machado BH. Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats. Am J Physiol Regul Integr Comp Physiol 302: R785-R793, 2012. First published December 28, 2011; doi:10.1152/ajpregu.00363.2011.-Sympathetic overactivity and altered respiratory control are commonly observed after chronic intermittent hypoxia (CIH) exposure. However, the central mechanisms underlying such neurovegetative dysfunctions remain unclear. Herein, we hypothesized that CIH (6% O-2 every 9 min, 8 h/day, 10 days) in juvenile rats alters glutamatergic transmission in the commissural nucleus tractus solitarius (cNTS), a pivotal site for integration of peripheral chemoreceptor inputs. Using an in situ working heart-brain stem preparation, we found that L-glutamate microinjections (1, 3, and 10 mM) into the cNTS of control rats (n = 8) evoked increases in thoracic sympathetic nerve (tSN) and central vagus nerve (cVN) activities combined with inhibition of phrenic nerve (PN) activity. Besides, the ionotropic glutamatergic receptor antagonism with kynurenic acid (KYN; 250 mM) in the cNTS of control group (n = 7) increased PN burst duration and frequency. In the CIH group (n = 10), the magnitude of L-glutamate-induced cVN excitation was smaller, and the PN inhibitory response was blunted (P < 0.05). In addition, KYN microinjections into the cNTS of CIH rats (n = 9) did not alter PN burst duration and produced smaller increases in its frequency compared with controls. Moreover, KYN microinjections into the cNTS attenuated the sympathoexcitatory response to peripheral chemoreflex activation in control but not in CIH rats (P < 0.05). These functional CIH-induced alterations were accompanied by a significant 10% increase of N-methyl-D-aspartate receptor 1 (NMDAR1) and glutamate receptor 2/3 (GluR2/3) receptor subunit density in the cNTS (n = 3-8, P < 0.05), evaluated by Western blot analysis. These data indicate that glutamatergic transmission is altered in the cNTS of CIH rats and may contribute to the sympathetic and respiratory changes observed in this experimental model.

Mechanisms of blunted muscle vasodilation during peripheral chemoreceptor stimulation in heart failure patients

We described recently that systemic hypoxia provokes vasoconstriction in heart failure (HF) patients. We hypothesized that either the exaggerated muscle sympathetic nerve activity and/or endothelial dysfunction mediate the blunted vasodilatation during hypoxia in HF patients. Twenty-seven HF patients and 23 age-matched controls were studied. Muscle sympathetic nerve activity was assessed by microneurography and forearm blood flow (FBF) by venous occlusion plethysmography. Peripheral chemoreflex control was evaluated through the inhaling of a hypoxic gas mixture (10% O-2 and 90% N-2). Basal muscle sympathetic nerve activity was greater and basal FBF was lower in HF patients versus controls. During hypoxia, muscle sympathetic nerve activity responses were greater in HF patients, and forearm vasodilatation in HF was blunted versus controls. Phentolamine increased FBF responses in both groups, but the increase was lower in HF patients. Phentolamine and N-G-monomethyl-L-arginine infusion did not change FBF responses in HF but markedly blunted the vasodilatation in controls. FBF responses to hypoxia in the presence of vitamin C were unchanged and remained lower in HF patients versus controls. In conclusion, muscle vasoconstriction in response to hypoxia in HF patients is attributed to exaggerated reflex sympathetic nerve activation and blunted endothelial function (NO activity). We were unable to identify a role for oxidative stress in these studies. (Hypertension. 2012; 60: 669-676.) . Online Data Supplement

Sympathoexcitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Botzinger complex of rats

Moraes DJ, Zoccal DB, Machado BH. Sympathoexcitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Botzinger complex of rats. J Neurophysiol 108: 610-623, 2012. First published April 25, 2012; doi:10.1152/jn.00057.2012.-The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Botzinger/pre-Botzinger complexes (RVLM/BotC/pre-BotC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BotC/pre-BotC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BotC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 +/- 0.02 vs. 1 +/- 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 +/- 2 vs. 82 +/- 2%; P < 0.05; n = 8), but decreased postinspiratory (35 +/- 3 vs. 12 +/- 2%; P < 0.05) and late-expiratory (24 +/- 4 vs. 2 +/- 1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 +/- 6 vs. 40 +/- 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 +/- 8 vs. 26 +/- 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BotC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BotC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BotC and RVLM/pre-BtC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BotC is essential to the coupling of active expiration and sympathoexcitatory response.

Microneurography in rats: a minimally invasive method to record single C-fiber action potentials from peripheral nerves in vivo

Microneurography is a method suitable for recording intraneural single or multiunit action potentials in conscious subjects. Microneurography has rarely been applied to animal experiments, where more invasive methods, like the teased fiber recording technique, are widely used. We have tested the feasibility of microneurographic recordings from the peripheral nerves of rats. Tungsten microelectrodes were inserted into the sciatic nerve at mid-thigh level. Single or multiunit action potentials evoked by regular electrical stimulation were recorded, digitized and displayed as a raster plot of latencies. The method allows unambiguous recording and recognition of single C-fiber action potentials from an in vivo preparation, with minimal disruption of the nerve being recorded. Multiple C-fibers can be recorded simultaneously for several hours, and if the animal is allowed to recover, repeated recording sessions can be obtained from the same nerve at the same level over a period of weeks or months. Also, single C units can be functionally identified by their changes in latency to natural stimuli, and insensitive units can be recognized as 'silent' nociceptors or sympathetic efferents by their distinctive profiles of activity-dependent slowing during repetitive electrical stimulation, or by the effect on spontaneous efferent activity of a proximal anesthetic block. Moreover, information about the biophysical properties of C axons can be obtained from their latency recovery cycles. Finally, we show that this preparation is potentially suitable for the study of C-fiber behavior in models of neuropathies and nerve lesions, both under resting conditions and in response to drug administration.

Plexiform hybrid granular cell tumor/perineurioma: a novel variant of benign peripheral nerve sheath tumor with divergent differentiation

The descriptive term hybrid peripheral nerve sheath tumor refers to any neoplasm of the neurilemmal apparatus composed of more than one pathologically defined tumoral equivalent derived from its constituent cells. Within this uncommon nosological category, participation of granular cell tumor - a neoplasm of modified Schwann cells - has been reported only exceptionally. We describe a hitherto not documented variant composed of an organoid mixture of granular cell tumor and perineurioma with plexiform growth. A solitary subcutaneous nodule of 1.5 cm diameter was excised from the right ring finger of a 19-year-old female with no antecedents of neurofibromatosis or relevant trauma. Histology revealed a monotonous, yet cytologically dimorphic proliferation of classical granular cells intermingled with flattened, inconspicuous perineurial cells. Immunohistochemical double labeling detected expression of S100 protein in the former and of EMA and GLUT-1 in the latter. While the respective staining patterns for S100 protein and EMA or GLUT-1 tended to be mutually exclusive, a minority of cells exhibited transitional granular cell/perineurial immunophenotype. Electron microscopy permitted direct visualization of a plethora of lysosomes in the granular cell moiety, and of pinocytotic vesicles and tight junctions in perineurial cells. Intratumoral axons were not detected. Expanding intraneurally, the lesion showed discrete encapsulation by the local perineurium, and resulted in plexiform growth. The MIB-1 labeling index averaged 1%. We interpret our findings as supporting evidence for the dual cell lineage to have arisen through metaplasia, with the tumor's dynamics probably having been driven by the granular cell component.

Acute alcohol ingestion and sympathetic neural responses during orthostatic stress in humans

Acute alcohol consumption has been reported to decrease mean arterial pressure (MAP) during orthostatic challenge, a response that may contribute to alcohol-mediated hypotension and eventually syncope. Muscle sympathetic nerve activity (MSNA) increases during orthostatic stress to help maintain MAP, yet the influence of alcohol on MSNA during orthostatic stress has not been determined. We hypothesized that alcohol ingestion would blunt arterial blood pressure and MSNA responses to progressive lower body negative pressure (LBNP). MAP, MSNA, and heart rate (HR) were recorded during progressive LBNP (-5, -10, -15, -20, -30, and -40 mmHg; 3 min/stage) in 30 subjects(age 24 ± 1 yrs). After an initial progressive LBNP protocol (pre-treatment), subjects were randomly assigned to consume alcohol (0.8g ethanol/kg body mass; n=15) or placebo (n=15) and then repeated the progressive LBNP protocol (post-treatment). Alcohol increased (drug × treatment, P ≤ 0.05) resting HR (59 ± 2 to 65 ± 2 beats/min) and MSNA (13 ± 3 to 19 ± 4 bursts/min) when compared to placebo. While alcohol increased MAP (83 ± 2 to 87 ± 2 mmHg), these increases were also observed with placebo (82 ± 2 to 88 ± 1 mmHg; treatment, P < 0.05; drug × treatment, P > 0.05). During progressive LBNP, a prominent decrease in MAP was observed after alcohol (drug × time × treatment, P < 0.05), but not placebo. There was also a significant attenuated response in forearm vascular resistance (FVR) during progressive LBNP (drug × time × treatment, P < 0.05). MSNA and HR increased during all LBNP protocols, but there were no differences between treatments or groups (drugs). In summary, acute alcohol ingestion induces an attenuation in blood pressure response during an orthostatic challenge, possibly due to the effect that alcohol has on impairing peripheral blood vessel constriction.

Nerve growth factor inhibits sympathetic neurons’ response to an injury cytokine

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phox gene in human neutrophils, monocytes, and B lymphocytes.

Antioxidant Defenses in the Peripheral Cell Layers of Legume Root Nodules1

Ascorbate peroxidase (AP) is a key enzyme that scavenges potentially harmful H2O2 and thus prevents oxidative damage in plants, especially in N2-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago sativa) root nodules contains elevated levels of AP protein, as well as the corresponding mRNA transcript and substrate (ascorbate). Enhanced AP protein levels were also found in cells immediately peripheral to the infected region of soybean (Glycine max), pea (Pisum sativum), clover (Trifolium pratense), and common bean (Phaseolus vulgaris) nodules. Regeneration of ascorbate was achieved by (homo)glutathione and associated enzymes of the ascorbate-glutathione pathway, which were present at high levels. The presence of high levels of antioxidants suggests that respiratory consumption of O2 in the endodermis or nodule parenchyma may be an essential component of the O2-diffusion barrier that regulates the entry of O2 into the central region of nodules and ensures optimal functioning of nitrogenase.