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.

Intermittent hypoxia-induced sensitization of central chemoreceptors contributes to sympathetic nerve activity during late expiration in rats

Molkov YI, Zoccal DB, Moraes DJ, Paton JF, Machado BH, Rybak IA. Intermittent hypoxia-induced sensitization of central chemoreceptors contributes to sympathetic nerve activity during late expiration in rats. J Neurophysiol 105: 3080-3091, 2011. First published April 6, 2011; doi:10.1152/jn.00070.2011.-Hypertension elicited by chronic intermittent hypoxia (CIH) is associated with elevated activity of the thoracic sympathetic nerve (tSN) that exhibits an enhanced respiratory modulation reflecting a strengthened interaction between respiratory and sympathetic networks within the brain stem. Expiration is a passive process except for special metabolic conditions such as hypercapnia, when it becomes active through phasic excitation of abdominal motor nerves (AbN) in late expiration. An increase in CO(2) evokes late-expiratory (late-E) discharges phase-locked to phrenic bursts with the frequency increasing quantally as hypercapnia increases. In rats exposed to CIH, the late-E discharges synchronized in AbN and tSN emerge in normocapnia. To elucidate the possible neural mechanisms underlying these phenomena, we extended our computational model of the brain stem respiratory network by incorporating a population of presympathetic neurons in the rostral ventrolateral medulla that received inputs from the pons, medullary respiratory compartments, and retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG). Our simulations proposed that CIH conditioning increases the CO(2) sensitivity of RTN/pFRG neurons, causing a reduction in both the CO(2) threshold for emerging the late-E activity in AbN and tSN and the hypocapnic threshold for apnea. Using the in situ rat preparation, we have confirmed that CIH-conditioned rats under normal conditions exhibit synchronized late-E discharges in AbN and tSN similar to those observed in control rats during hypercapnia. Moreover, the hypocapnic threshold for apnea was significantly lowered in CIH-conditioned rats relative to that in control rats. We conclude that CIH may sensitize central chemoreception and that this significantly contributes to the neural impetus for generation of sympathetic activity and hypertension.

Modulation of respiratory responses to chemoreflex activation by L-glutamate and ATP in the rostral ventrolateral medulla of awake rats

Moraes DJA, Bonagamba LGH, Zoccal DB, Machado BH. Modulation of respiratory responses to chemoreflex activation by L-glutamate and ATP in the rostral ventrolateral medulla of awake rats. Am J Physiol Regul Integr Comp Physiol 300: R1476-R1486, 2011. First published March 16, 2011; doi:10.1152/ajpregu.00825.2010.-Presympathetic neurons in the different anteroposterior aspects of rostral ventrolateral medulla (RVLM) are colocalized with expiratory [Botzinger complex (BotC)] and inspiratory [pre-Botzinger complex (pre-BotC)] neurons of ventral respiratory column (VRC), suggesting that this region integrates the cardiovascular and respiratory chemoreflex responses. In the present study, we evaluated in different anteroposterior aspects of RVLM of awake rats the role of ionotropic glutamate and purinergic receptors on cardiorespiratory responses to chemoreflex activation. The bilateral ionotropic glutamate receptors antagonism with kynurenic acid (KYN) (8 nmol/50 nl) in the rostral aspect of RVLM (RVLM/BotC) enhanced the tachypneic (120 +/- 9 vs. 180 +/- 9 cpm; P < 0.01) and attenuated the pressor response (55 +/- 2 vs. 15 +/- 1 mmHg; P < 0.001) to chemoreflex activation (n = 7). On the other hand, bilateral microinjection of KYN into the caudal aspect of RVLM (RVLM/pre-BotC) caused a respiratory arrest in four awake rats used in the present study. Bilateral P2X receptors antagonism with PPADS (0.25 nmol/50 nl) in the RVLM/BotC reduced chemoreflex tachypneic response (127 +/- 6 vs. 70 +/- 5 cpm; P < 0.001; n = 6), but did not change the chemoreflex pressor response. In addition, PPADS into the RVLM/BtC attenuated the enhancement of the tachypneic response to chemoreflex activation elicited by previous microinjections of KYN into the same subregion (188 +/- 2 vs. 157 +/- 3 cpm; P < 0.05; n = 5). Our findings indicate that: 1) L-glutamate, but not ATP, in the RVLM/BtC is required for pressor response to peripheral chemoreflex and 2) both transmitters in the RVLM/BtC are required for the processing of the ventilatory response to peripheral chemoreflex activation in awake rats.

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O(2) for 40 s, every 9 min, 8 h day(-1)) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n = 14) exhibited higher levels of mean arterial pressure than controls (101 +/- 3 versus 89 +/- 3 mmHg, n = 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart-brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 +/- 5 versus 40 +/- 3%; n = 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post-inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic-respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Short-term sustained hypoxia induces changes in the coupling of sympathetic and respiratory activities in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Physiological and pathophysiological interactions between the respiratory central pattern generator and the sympathetic nervous system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of ventral surface CO2/H+-sensitive neurons by purinergic signalling

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+. Abrainstemregion called the retrotrapezoid nucleus (RTN) contains a population of CO2/H+-sensitive neurons that appears to function as an important chemoreceptor. Evidence also indicates that CO2-evoked ATP release from RTN astrocytes modulates activity of CO2/H+-sensitive neurons; however, the extent to which purinergic signalling contributes to chemoreception by RTN neurons is not clear and the mechanism(s) underlying CO2/H+-evoked ATP release is not fully elucidated. The goals of this study are to determine the extent to which ATP contributes to RTN chemoreception both in vivo and in vitro, andwhether purinergic drive to chemoreceptors relies on extracellularCa(2+) or gap junction hemichannels. We also examine the possible contribution of P2Y1 receptors expressed in theRTNto the purinergic drive to breathe. We showthat purinergic signalling contributes, in part, to the CO2/H+ sensitivity of RTN neurons. In vivo, phrenic nerve recordings of respiratory activity in adult rats show that bilateral injections of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2 receptor blocker) decreased the ventilatory response to CO2 by 30%. In vitro, loose-patch recordings from RTN neurons show that P2 receptor blockers decreased responsiveness to both 10% and 15% CO2 also by 30%. In the slice, the contribution of purinergic signalling to RTN chemoreception did not increase with temperature (22-35 degrees C) and was retained in low extracellular Ca2+ medium. Conversely, the gap junction blockers carbenoxolone and cobalt decreased neuronal CO2/H+ sensitivity by an amount similar to P2 receptor antagonists. Inhibition of the P2Y1 receptor in the RTN had no effect on CO2 responsivness in vitro or in vivo; thus, the identity of P2 receptors underlying the purinergic component of RTN chemoreception remains unknown. These results support the possibility that CO2/H+-evoked ATP release is mediated by a mechanism involving gap junction hemichannels.

Pontomedullary and hypothalamic distribution of Fos-like immunoreactive neurons after acute exercise in rats

During exercise, intense brain activity orchestrates an increase in muscle tension. Additionally, there is an increase in cardiac output and ventilation to compensate the increased metabolic demand of muscle activity and to facilitate the removal of CO2 from and the delivery of O-2 to tissues. Here we tested the hypothesis that a subset of pontomedullary and hypothalamic neurons could be activated during dynamic acute exercise. Male Wistar rats (250-350 g) were divided into an exercise group (n = 12) that ran on a treadmill and a no-exercise group (n = 7). Immunohistochemistry of pontomedullary and hypothalamic sections to identify activation (c-Fos expression) of cardiorespiratory areas showed that the no-exercise rats exhibited minimal Fos expression. In contrast, there was intense activation of the nucleus of the solitary tract, the ventrolateral medulla (including the presumed central chemoreceptor neurons in the retrotrapezoid/parafacial region), the lateral parabrachial nucleus, the Kolliker-Fuse region, the perifornical region, which includes the perifornical area and the lateral hypothalamus, the dorsal medial hypothalamus, and the paraventricular nucleus of the hypothalamus after running exercise. Additionally, we observed Fos immunoreactivity in catecholaminergic neurons within the ventrolateral medulla (C1 region) without Fos expression in the A2, A5 and A7 neurons. In summary, we show for the first time that after acute exercise there is an intense activation of brain areas crucial for cardiorespiratory control. Possible involvement of the central command mechanism should be considered. Our results suggest whole brain-specific mobilization to correct and compensate the homeostatic changes produced by acute exercise. (c) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Opioid mu-receptors in the rostral medullary raphe modulate hypoxia-induced hyperpnea in unanesthetized rats

Aim: It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As opioid mu-receptors have been found in the MR, we studied the putative role of opioid mu-receptors in the rostral MR (rMR) region on ventilation in normal and 7% hypoxic conditions. Methods: We measured pulmonary ventilation ((V) over dotE) and the body temperatures (Tb) of male Wistar rats before and after the selective opioid l-receptor antagonist CTAP ( d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2, cyclic, 0.1 mu g per 0.1 mu L) was microinjected into the rMR during normoxia or after 60 min of hypoxia. Results: The animals treated with intra-rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia ( 430 +/- 86 mL kg) 1 min) 1) compared with the control group ( 790 +/- 82 mL kg) 1 min) 1) ( P < 0.05). No differences in the Tb were observed between groups during hypoxia. Conclusion: These data suggest that opioids acting on l-receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia.

Contribution of the retrotrapezoid nucleus/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats

Moraes DJ, Dias MB, Cavalcanti-Kwiatkoski R, Machado BH, Zoccal DB. Contribution of retrotrapezoid nucleus/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats. J Neurophysiol 108: 882-890, 2012. First published May 16, 2012; doi:10.1152/jn.00193.2012.-Central mechanisms of coupling between respiratory and sympathetic systems are essential for the entrainment between the enhanced respiratory drive and sympathoexcitation in response to hypoxia. However, the brainstem nuclei and neuronal network involved in these respiratory-sympathetic interactions remain unclear. Here, we evaluated whether the increase in expiratory activity and expiratory-modulated sympathoexcitation produced by the peripheral chemoreflex activation involves the retrotrapezoid nucleus/parafacial respiratory region (RTN/pFRG). Using decerebrated arterially perfused in situ rat preparations (60-80 g), we recorded the activities of thoracic sympathetic (tSN), phrenic (PN), and abdominal nerves (AbN) as well as the extracellular activity of RTN/pFRG expiratory neurons, and reflex responses to chemoreflex activation were evaluated before and after inactivation of the RTN/pFRG region with muscimol (1 mM). In the RTN/pFRG, we identified late-expiratory (late-E) neurons (n = 5) that were silent at resting but fired coincidently with the emergence of late-E bursts in AbN after peripheral chemoreceptor activation. Bilateral muscimol microinjections into the RTN/pFRG region (n = 6) significantly reduced basal PN frequency, mean AbN activity, and the amplitude of respiratory modulation of tSN (P < 0.05). With respect to peripheral chemoreflex responses, muscimol microinjections in the RTN/pFRG enhanced the PN inspiratory response, abolished the evoked late-E activity of AbN, but did not alter either the magnitude or pattern of the tSN reflex response. These findings indicate that the RTN/pFRG region is critically involved in the processing of the active expiratory response but not of the expiratory-modulated sympathetic response to peripheral chemoreflex activation of rat in situ preparations.

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

A New Platinum Complex With Tryptophan: Synthesis, Structural Characterization, Dft Studies And Biological Assays In Vitro Over Human Tumorigenic Cells.

A new platinum(II) complex with the amino acid L-tryptophan (trp), named Pt-trp, was synthesized and characterized. Elemental, thermogravimetric and ESI-QTOF mass spectrometric analyses led to the composition [Pt(C11H11N2O2)2]⋅6H2O. Infrared spectroscopic data indicate the coordination of trp to Pt(II) through the oxygen of the carboxylate group and also through the nitrogen atom of the amino group. The (13)C CP/MAS NMR spectroscopic data confirm coordination through the oxygen atom of the carboxylate group, while the (15)N CP/MAS NMR data confirm coordination of the nitrogen of the NH2 group to the metal. Density functional theory (DFT) studies were applied to evaluate the cis and trans coordination modes of trp to platinum(II). The trans isomer was shown to be energetically more stable than the cis one. The Pt-trp complex was evaluated as a cytotoxic agent against SK-Mel 103 (human melanoma) and Panc-1 (human pancreatic carcinoma) cell lines. The complex was shown to be cytotoxic over the considered cells.