Plasticity of cardiovascular chemoreflexes after prolonged unilateral carotid body denervation: implications for its therapeutic use.

Unilateral carotid body denervation has been proposed as treatment for sympathetic-related human diseases such as systolic heart failure, hypertension, obstructive sleep apnea, and cardiometabolic diseases. The long-term therapeutic effects of carotid body removal will be maintained if the remnant "buffer nerves," that is, the contralateral carotid nerve and the aortic nerves that innervate second-order neurons at the solitary tract nuclei (NTS), do not modify their contributions to the cardiovascular chemoreflexes. Here, we studied the cardiovascular chemoreflexes 1 mo after unilateral carotid body denervation either by excision of the petrosal ganglion (petrosal ganglionectomy, which eliminates central carotid afferents) or exeresis of a segment of one carotid nerve (carotid neurectomy, which preserves central afferents). Cardiovascular chemoreflexes were induced by intravenous (iv) injections of sodium cyanide in pentobarbitone-anesthetized adult cats. After 1 mo of unilateral petrosal ganglionectomy, without significant changes in basal arterial pressure, the contribution of the contralateral carotid nerve to the chemoreflex increases in arterial pressure was enhanced without changes in the contribution provided by the aortic nerves. By contrast, after 1 mo of unilateral carotid neurectomy, the contribution of remnant buffer nerves to cardiovascular chemoreflexes remained unmodified. These results indicate that a carotid nerve interruption involving denervation of second-order chemosensory neurons at the NTS will trigger cardiovascular chemoreflex plasticity on the contralateral carotid pathway. Then, unilateral carotid body denervation as therapeutic tool should consider the maintenance of the integrity of carotid central chemoafferents to prevent plasticity on remnant buffer nerves.NEW & NOTEWORTHY Unilateral carotid body denervation has been proposed as treatment for sympathetic hyperactivity-related human disorders. Its therapeutic effectiveness for maintaining a persistent decrease in the sympathetic outflow activity will depend on the absence of compensatory chemoreflex plasticity in the remnant carotid and aortic afferents. Here, we suggest that the integrity of central afferents after carotid body denervation is essential to prevent the emergence of plastic functional changes on the contralateral "intact" carotid nerve.

BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation.

Nitric oxide in the commissural nucleus tractus solitarii regulates carotid chemoreception hyperglycemic reflex and c-Fos expression.

Carotid body chemoreceptors function as glucose sensors and contribute to glucose homeostasis. The nucleus tractus solitarii (NTS) is the first central nervous system (CNS) nuclei for processing of information arising in the carotid body. Here, we microinjected a nitric oxide (NO) donor sodium nitroprusside (SNP), an NO-independent activator of the soluble guanylyl cyclase (sGC) (YC1) or an NO-synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) into the commissural NTS (cNTS) before carotid chemoreceptor anoxic stimulation and measured arterial glucose and the expression of Fos-like immunoreactivity (Fos-ir). Male Wistar rats (250-300 g) were anesthetized, and the carotid sinus was vascularly isolated. Either artificial cerebrospinal fluid (aCSF), SNP, YC1 or L-NAME were stereotaxically injected into the cNTS. The SNP and YC1 infused into the cNTS before carotid chemoreceptor stimulation (SNP-2 and YC1-2 groups) similarly increased arterial glucose compared to the aCSF-2 group. By contrast, infusion of L-NAME into the cNTS before carotid chemoreceptor stimulation (L-NAME-2 group) decreased arterial glucose concentration. The number of cNTS Fos-ir neurons, determined in all the groups studied except for YC1 groups, significantly increased in SNP-2 rat when compared to the aCSF-2 or SNP-2 groups. Our findings demonstrate that NO signaling, and the correlative activation of groups of cNTS neurons, plays key roles in the hyperglycemic reflex initiated by carotid chemoreceptor stimulation.

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.(AU)

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Branchial O(2) chemoreceptors in Nile tilapia Oreochromis niloticus: Control of cardiorespiratory function in response to hypoxia.

This study examined the distribution and orientation of gill O(2) chemoreceptors in Oreochromis niloticus and their role in cardiorespiratory responses to graded hypoxia. Intact fish, and a group with the first gill arch excised (operated), were submitted to graded hypoxia and their cardiorespiratory responses (oxygen uptake - V˙O(2) , breathing frequency - fR, ventilatory stroke volume - VT, gill ventilation - V˙G, O(2) extraction from the ventilatory current - EO(2) , and heart rate - fH) were compared. Their responses to bolus injections of NaCN into the bloodstream (internal) or ventilatory water stream (external) were also determined. The V˙O(2) of operated fish was significantly lower at the deepest levels of hypoxia. Neither reflex bradycardia nor ventilatory responses were completely abolished by bilateral excision of the first gill arch. EO(2) of the operated group was consistently lower than the intact group. The responses to internal and external NaCN included transient decreases in fH and increases in fR and Vamp (ventilation amplitude). These cardiorespiratory responses were attenuated but not abolished in the operated group, indicating that chemoreceptors are not restricted to the first gill arch, and are sensitive to oxygen levels in both blood and water.

Brain-derived neurotrophic factor in the nucleus tractus solitarii modulates glucose homeostasis after carotid chemoreceptor stimulation in rats.

Neuronal systems, which regulate energy intake, energy expenditure and endogenous glucose production, sense and respond to input from hormonal related signals that convey information from body energy availability. Carotid chemoreceptors (CChr) function as sensors for circulating glucose levels and contribute to glycemic counterregulatory responses. Brain-derived neurotrophic factor (BDNF) that plays an important role in the endocrine system to regulate glucose metabolism could play a role in hyperglycemic glucose reflex with brain glucose retention (BGR) evoked by anoxic CChr stimulation. Infusing BDNF into the nucleus tractus solitarii (NTS) before CChr stimulation, showed that this neurotrophin increased arterial glucose and BGR. In contrast, BDNF receptor (TrkB) antagonist (K252a) infusions in NTS resulted in a decrease in both glucose variables.

Nitric oxide infused in the solitary tract nucleus blocks brain glucose retention induced by carotid chemoreceptor stimulation.

Previous work has shown that the carotid body glomus cells can function as glucose sensors. The activation of these chemoreceptors, and of its afferent nucleus in the brainstem (solitary tract nucleus - STn), induces rapid changes in blood glucose levels and brain glucose retention. Nitric oxide (NO) in STn has been suggested to play a key role in the processing of baroreceptor signaling initiated in the carotid sinus. However, the relationship between changes in NO in STn and carotid body induced glycemic changes has not been studied. Here we investigated in anesthetized rats how changes in brain glucose retention, induced by the local stimulation of carotid body chemoreceptors with sodium cyanide (NaCN), were affected by modulation of NO levels in STn. We found that NO donor sodium nitroprusside (SNP) micro-injected into STn completely blocked the brain glucose retention reflex induced by NaCN chemoreceptor stimulation. In contrast, NOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) increased brain glucose retention reflex compared to controls or to SNP rats. Interestingly, carotid body stimulation doubled the expression of nNOS in STn, but had no effect in iNOS. NO in STn could function to terminate brain glucose retention induced by carotid body stimulation. The work indicates that NO and STn play key roles in the regulation of brain glucose retention.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats.

A rise in arterial Pco(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Bötzinger/C1 region (Bötz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 ± 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Δ = +56 ± 8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Bötz/C1 reduced PND (Δ = +43 ± 7%, +52 ± 6% or +56 ± 11%, respectively). MCPG also reduced sSND (Δ = +41 ± 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Bötz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Bötz/C1.

Hypoxic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): role of branchial O2 chemoreceptors.

In one series of experiments, heart frequency (f (H)), blood pressure (P (a)), gill ventilation frequency (f ( R )), ventilation amplitude (V (AMP)) and total gill ventilation (V (TOT)) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO(2) approximately 140, normoxia to 17 mmHg, severe hypoxia), they increased f ( R ), V (AMP), V (TOT) and P (a) and decreased f (H). In a second series of experiments, air-breathing frequency (f (RA)), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 microg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 microg NaCN in 1.0 mL water) on f (R), V (AMP), V (TOT), P (a) and f (H) we conclude that the O(2) receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O(2) levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.

Nitric oxide in the hypothalamus-pituitary axis mediates increases in brain glucose retention induced by carotid chemoreceptor stimulation with cyanide in rats.

Neuronal nitric oxide synthase (nNOS), which catalyzes the generation of nitric oxide (NO), is expressed by neuron subpopulations in the CNS. Nitric oxide is involved in neurotransmission and central glucose homeostasis. Our prior studies have shown that carotid body receptors participate in brain glucose regulation in vivo, and suggest the presence of a NO tonic mechanism in the solitary tract nucleus (STn). However, the role of NO within STn in glucose control remains unknown. In this study, we explored the potential regulatory role of NO on brain glucose retention induced by carotid body chemoreceptor anoxic stimulation with sodium cyanide (NaCN) which inhibits oxidative metabolism. Intracisternal infusions of nitroxidergic drugs before carotid chemoreceptor stimulation in anesthetized rats, elicited changes in nitrite concentration in plasma and hypothalamus-pituitary (H-P) tissue, as well as in gene expression of neuronal and inducible isoforms (nNOS and iNOS) in H-P tissue. The changes observed in above variables modified brain glucose retention in an opposite direction. When the NO donor, sodium nitroprusside (SNP), was given before carotid stimulation, nitrite concentration in plasma and H-P tissue, and gene expression of nNOS and iNOS in H-P tissue increased, whereas brain glucose retention decreased. In contrast, when the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME) was infused immediately before carotid chemoreceptor stimulation, nitrite levels and nNOS expression decreased in plasma and H-P tissue, whereas brain glucose retention increased. Anoxic stimulation by itself induced an increase in the expression of both genes studied. All these results indicate that de novo expression of the nNOS gene in H-P tissue may be critically involved in central glucose changes observed after anoxic carotid chemoreceptor stimulation in conjunction with NO.

The relationship between O2 chemoreceptors, cardio-respiratory reflex and hypoxia tolerance in the neotropical fish Hoplias lacerdae.

The localization, distribution and orientation of O(2) chemoreceptors associated with the control of cardio-respiratory responses were investigated in the neotropical, Hoplias lacerdae. Selective denervation of the cranial nerves (IX and X) was combined with chemical stimulation (NaCN) to characterize the gill O(2) chemoreceptors, and the fish were then exposed to gradual hypoxia to examine the extent of each cardio-respiratory response. Changes in heart rate (f(H)) and ventilation amplitude (V(amp)) were allied with chemoreceptors distributed on both internal and external surfaces of all gill arches, while ventilation rate (f) was allied to the O(2) chemoreceptors located only in the internal surface of the first gill arch. H. lacerdae exposed to gradual hypoxia produced a marked bradycardia (45%) and 50% increase in V(amp), but only a relatively small change in f (32%). Thus, the low f(R) response yet high V(amp) were in accord with the characterization of the O(2) chemoreceptors. Comparing these results from H. lacerdae with hypoxia-tolerant species revealed a relationship existent between general oxygenation of the individual species environment, its cardio-respiratory response to hypoxia and the characterization of O(2) chemoreceptors.

Nitric oxide in the solitary tract nucleus (STn) modulates glucose homeostasis and FOS-ir expression after carotid chemoreceptor stimulation.

We evaluate in rats the role of NO in the solitary tract nucleus (STn) after an anoxic stimulus to carotid body chemoreceptor cells (CChrc) with cyanide (NaCN), on the hyperglycemic reflex with glucose retention by the brain (BGR) and FOS expression (FOS-ir) in the STn. The results suggest that nitroxidergic pathways in the STn may play an important role in glucose homeostasis. A NO donor such as sodium nitroprusside (NPS) in the STn before CChrc stimulation increased arterial glucose level and significantly decreased BGR. NPS also induced a higher FOS-ir expression in STn neurons when compared to neurons in control rats that only received artificial cerebrospinal fluid (aCSF) before CChrc stimulation. In contrast, a selective NOS inhibitor such as Nomega-nitro-L-arginine methyl ester (L-NAME) in the STn before CChrc stimulation resulted in an increase of both, systemic glucose and BGR above control values. In this case, the number of FOS-ir positive neurons in the STn decreased when compared to control or to NPS experiments. FOS-ir expression in brainstem cells suggests that CChrc stimulation activates nitroxidergic pathways in the STn to regulate peripheral and central glucose homeostasis. The study of these functionally defined cells will be important to understand brain glucose homeostasis.

Gill chemoreceptors and cardio-respiratory reflexes in the neotropical teleost pacu, Piaractus mesopotamicus.

This study examined the location and distribution of O(2) chemoreceptors involved in cardio-respiratory responses to hypoxia in the neotropical teleost, the pacu (Piaractus mesopotamicus). Intact fish and fish experiencing progressive gill denervation by selective transection of cranial nerves IX and X were exposed to gradual hypoxia and submitted to intrabuccal and intravenous injections of NaCN while their heart rate, ventilation rate and ventilation amplitude were measured. The chemoreceptors producing reflex bradycardia were confined to, but distributed along all gill arches, and were sensitive to O(2) levels in the water and the blood. Ventilatory responses to all stimuli, though modified, continued following gill denervation, however, indicating the presence of internally and externally oriented receptors along all gill arches and either in the pseudobranch or at extra-branchial sites. Chemoreceptors located on the first pair of gill arches and innervated by the glossopharyngeal nerve appeared to attenuate the cardiac and respiratory responses to hypoxia. The data indicate that the location and distribution of cardio-respiratory O(2) receptors are not identical to those in tambaqui (Colossoma macropomum) despite their similar habitats and close phylogenetic lineage, although the differences between the two species could reduce to nothing more than the presence or absence of the pseudobranch.

Effects of intermittent hypoxia on cat petrosal ganglion responses induced by acetylcholine, adenosine 5'-triphosphate and NaCN.

Exposure to chronic intermittent hypoxia (CIH) for 4 days enhances the cat carotid body (CB) chemosensory responses to acute hypoxia. However, it is not known if CIH enhances the responses of the petrosal ganglion (PG) neurons that innervate the CB chemoreceptor cells. Accordingly, we studied the effects of the CB putative excitatory transmitter acetylcholine (ACh) and adenosine 5 -triphosphate (ATP), and the effects of citotoxic hypoxia (NaCN) applied to the isolated PG from cats exposed to CIH for 4 days. The dose-dependent curve parameters of the frequency of discharges evoked in the carotid sinus nerve by the application of ACh, ATP and NaCN to the isolated PG in control condition were not significantly modified in the CIH-treated cats. Present results suggest that CIH enhances the chemosensory responses to acute hypoxia acting primarily at the chemoreceptor cells, without major changes in the response of PG neurons evoked by the application of putative CB excitatory transmitters to their somata.

Papel del óxido nítrico en la retención de glucosa cerebral post-estimulación de los receptores carotídeos con cianuro de sodio en ratas / The role of the nitric oxide to retain the cerebral glucose post-stimulation of the carotid receptors with sodium cyanide in rats

Se realizó un estudio experimental, con el objetivo de medir la acción del óxido nítrico (NO) en la captación de glucosa cerebral, después de la estimulación con cianuro de sodio (NaCN-5µg/100g) de los receptores del cuerpo carotídeo (RCC). Los experimentos se realizaron en ratas (280-310g) anestesiadas, mantenidas con respiración artificial a una temperatura de 25°C. Los protocolos fueron el control I, la perfusión en cisterna magna (CM) de líquido cefalorraquídeo artificial-LCRa (5 µL/30 s), el control II, la ERC en forma simultánea con la perfusión de LCRa, la perfusión de un donador de NO (nitroglicerina) (NG-3µg/5µL de LCRa) en CM, la ERC en forma simultánea con NG en CM, la perfusión de un inhibidor de NO (L-NAME) (250µg/5µL de LCRa), la ERC en forma simultánea con L-NAME en CM. Los resultados obtenidos indican que la combinación de NG con ERC no altera la retención de glucosa cerebral, mientras que en los controles, la NG sola aumentó la retención cerebral de glucosa. Por el contrario, el L-NAME en combinación con ERC aumentó la captación de glucosa cerebral e indicó que el óxido nítrico desempeña un papel modulador en la respuesta hiperglucemiante en los estados de hipoxia

The relationship between the surface composition and electrical properties of corrosion films formed on carbon steel in alkaline sour medium: an XPS and EIS study.

This work studies the evolution of 1018 carbon steel surfaces during 3-15 day immersion in alkaline sour medium 0.1 M (NH4)2S and 10 ppm CN(-) as (NaCN). During this period of time, surfaces were jointly characterized by electrochemical techniques in situ (electrochemical impedance spectroscopy, EIS) and spectroscopic techniques ex situ (X-ray photoelectron spectroscopy, XPS). The results obtained by these techniques allowed for a description of electrical and chemical properties of the films of corrosion products formed at the 1018 steel surface. There is an interconversion cycle of chemical species that form films of corrosion products whose conversion reactions favor two different types of diffusions inside the films: a chemical diffusion of iron cations and a typical diffusion of atomic hydrogen. These phenomena jointly control the passivity of the interface attacked by the corrosive medium.

Carotid chemoreceptor reflex modulation by arginine-vasopressin microinjected into the nucleus tractus solitarius in rats.

BACKGROUND: In addition to their role of sensing O2, pH, CO2, osmolarity and temperature, carotid body receptors (CBR) were proposed by us and others to have a glucose-sensing role in the blood entering the brain, integrating information about blood glucose and O2 levels essential for central nervous system (CNS) metabolism. The nucleus tractus solitarius (NTS) is an important relay station in central metabolic control and receives signals from peripheral glucose-sensitive hepatoportal afferences, from central glucose-responsive neurons in the brainstem and from CBR and arginine-vasopressin (AVP)-containing axons from hypothalamic nuclei. METHODS: In normal Wistar rats anesthetized with pentobarbital, permanent cannulas were placed stereotaxically in the NTS. Glucose changes were induced in vivo after CBR stimulation with sodium cyanide (NaCN-5 microg/100 g), preceded by an infusion of AVP [(10 or 40 pmol/100 nL of artificial cerebrospinal fluid) aCSF] or an antagonist for V1a receptors (anti-glycogenolytic vasopressin analogue-VP1-A) (100 pmol/100 nL of aCSF) into the NTS. RESULTS: CBR stimulation after an AVP infusion (larger dose) into the NTS resulted in a significantly higher arterial glucose and lower brain arterial-venous glucose difference. In the same way, VP1-A administration in the NTS significantly decreased the effects observed after AVP priming before CBR stimulation or preceding the CBR stimulation, alone. CONCLUSIONS: We propose that AVP in the NTS could participate in glucose homeostasis, modulating the information arising in CBR after histotoxic-anoxia stimulation.

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite.

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.