ABSTRACT
AIM: Lung ischaemia-reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia-reperfusion-induced pulmonary vasoconstriction. The aim was to determine whether ischaemia-reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion. METHODS: Ischaemia-reperfusion was induced for various times in anesthetized rabbits with ventilated lungs by reversibly occluding the right pulmonary artery and initiating reperfusion. Nitric oxide synthase activity/expression and phosphorylation, reactive nitrogen species generation and total nitrate/nitrite were determined in lung tissue. RESULTS: Inducible nitric oxide synthase expression and activity, and reactive nitrogen species formation coincided with increased pulmonary vascular resistance during reperfusion and increased with ischaemia duration, further increasing after 2-h reperfusion. Total nitrate/nitrite also increased with ischaemia but decreased after 2-h reperfusion. Pre-treatment with an inducible nitric oxide synthase inhibitor (1400W; Cayman Chemical Company, Ann Arbor, MI, USA) attenuated inducible nitric oxide synthase activity, reactive nitrogen species generation and pulmonary vascular resistance, but did not affect total nitrate/nitrite. Endothelial nitric oxide synthase expression was unchanged by ischaemia-reperfusion; however, its phosphorylation on serine 1177 and dephosphorylation on threonine 495 was uncoupled, suggesting decreased endothelial nitric oxide synthase activity. 1400W prevented uncoupling of endothelial nitric oxide synthase phosphorylation, maintaining its activity during reperfusion. CONCLUSION: Ischaemia-reperfusion up-regulates inducible nitric oxide synthesis and/activity, which coincides with reduced endothelial nitric oxide synthase activity as suggested by its uncoupling and may contribute to ischaemia-reperfusion-induced pulmonary vasoconstriction.
Subject(s)
Lung/blood supply , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide/metabolism , Pulmonary Artery/enzymology , Reperfusion Injury/enzymology , Vasoconstriction , Animals , Disease Models, Animal , Enzyme Inhibitors/pharmacology , NADPH Oxidases/metabolism , Nitrates/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitrites/metabolism , Phosphorylation , Pulmonary Artery/drug effects , Pulmonary Artery/physiopathology , Pulmonary Circulation , Rabbits , Reactive Nitrogen Species/metabolism , Reperfusion Injury/physiopathology , Respiration, Artificial , Time Factors , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Vascular Resistance , Vasoconstriction/drug effectsABSTRACT
Intermittent hypoxia (IH) during sleep, a characteristic feature of sleep-disordered breathing (SDB) is associated with time-dependent apoptosis and spatial learning deficits in the adult rat. The mechanisms underlying such neurocognitive deficits remain unclear. Activation of the cAMP-response element binding protein (CREB) transcription factor mediates critical components of neuronal survival and memory consolidation in mammals. CREB phosphorylation and DNA binding, as well as the presence of apoptosis in the CA1 region of the hippocampus were examined in Sprague-Dawley male rats exposed to IH. Spatial reference task learning was assessed with the Morris water maze. IH induced significant decreases in Ser-133 phosphorylated CREB (pCREB) without changes in total CREB, starting as early as 1 h IH, peaking at 6 h-3 days, and returning toward normoxic levels by 14-30 days. Double-labeling immunohistochemistry for pCREB and Neu-N (a neuronal marker) confirmed these findings. The expression of cleaved caspase 3 (cC3) in the CA1, a marker of apoptosis, peaked at 3 days and returned to normoxic values at 14 days. Initial IH-induced impairments in spatial learning were followed by partial functional recovery starting at 14 days of IH exposure. We postulate that IH elicits time-dependent changes in CREB phosphorylation and nuclear binding that may account for decreased neuronal survival and spatial learning deficits in the adult rat. We suggest that CREB changes play an important role in the neurocognitive morbidity of SDB patients.
Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Hippocampus/metabolism , Hippocampus/radiation effects , Hypoxia/metabolism , Maze Learning/physiology , Photoperiod , Animals , Behavior, Animal , Blotting, Western , Caspase 3 , Caspases/metabolism , Enzyme-Linked Immunosorbent Assay , Escape Reaction , Hypoxia/physiopathology , Immunohistochemistry , Light , Male , Phosphorylation , Protein Binding , Rats , Rats, Sprague-Dawley , Swimming , Time FactorsABSTRACT
Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP >> ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.
Subject(s)
Adenosine Diphosphate/pharmacology , Macrophages, Alveolar/drug effects , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Respiratory Burst/drug effects , Animals , Enzyme Activation , Free Radicals , Genistein/pharmacology , Immunoblotting , Macrophages, Alveolar/metabolism , NADPH Oxidases/metabolism , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins c-akt , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species , Receptors, Purinergic/metabolism , Signal Transduction , Superoxides , Zymosan/pharmacologyABSTRACT
The temporal trajectory of platelet-derived growth factor (PDGF)-beta receptor activation within the dorsocaudal brainstem parallels that of the mild hypoxic ventilatory depression (HVD) seen in adult rats. We hypothesized that enhanced PDGF-beta receptor activity may account for the particularly prominent HVD of developing mammals. To study this issue, 2-, 5-, 10-, and 20-d-old rats underwent hypoxic challenges (10% O(2) for 30 min) after pretreatment with either vehicle (Veh) or the selective PDGF-beta receptor antagonist CGP57148B (intraperitoneal 100 mg/kg). The developmental characteristics and magnitude of the peak hypoxic ventilatory response (HVR) were not modified by the PDGF-beta receptor blocker. However, HVD was markedly attenuated by CGP57148B, and such effect, although still present, gradually abated with increasing postnatal age [p < 0.001, analysis of variance (ANOVA)]. Hypercapnic ventilatory responses were not affected by CGP57148B. The expression of PDGF-beta receptor in the dorsocaudal brainstem was assessed by immunoblotting and confirmed progressively decreasing expression with maturation. We conclude that PDGF-beta receptor activation during hypoxia is an important contributor to HVD at all postnatal ages but more particularly in the immature rat.
Subject(s)
Brain Stem/metabolism , Hypoxia/metabolism , Pulmonary Ventilation/physiology , Receptor, Platelet-Derived Growth Factor beta/metabolism , Animals , Benzamides , Brain Stem/drug effects , Brain Stem/growth & development , Hypercapnia/metabolism , Hypercapnia/physiopathology , Hypoxia/physiopathology , Imatinib Mesylate , Immunoblotting , Piperazines/pharmacology , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitorsABSTRACT
Ischemic preconditioning (PC) of heart and brain is a well-documented phenomenon. However, the mechanism underlying the increased resistance to severe ischemia by a preceding mild ischemic exposure remains unclear. Over a decade ago, we demonstrated the existence of hypoxic PC in the hippocampal slice preparation. Here we report the ability of a short exposure to toxic levels of glutamate to heighten the tolerance of hippocampal slices to a subsequent, longer exposure to the excitotoxin. Glutamate PC could also be induced by a short hypoxic exposure, suggesting a common mechanistic pathway for all PC stimuli. Since glutamate receptor activation and hypoxia increase tissue lactate production, a-cyano-4-hydroxycinnamate was applied during the PC period to completely abolished PC. These results indicate that excitotoxic PC and hypoxic PC share similar mechanisms that possibly involve lactate production and its neuronal utilization.
Subject(s)
Carrier Proteins/antagonists & inhibitors , Glutamic Acid/pharmacology , Hippocampus/drug effects , Hypoxia-Ischemia, Brain/metabolism , Ischemic Preconditioning/methods , Lactic Acid/metabolism , Neurotoxins/pharmacology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Carrier Proteins/metabolism , Coumaric Acids/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Glutamic Acid/metabolism , Hippocampus/metabolism , Hypoxia-Ischemia, Brain/physiopathology , Male , N-Methylaspartate/pharmacology , Neurons/drug effects , Neurons/metabolism , Organ Culture Techniques , Rats , Rats, Sprague-DawleyABSTRACT
Obstructive sleep apnea is characterized by intermittent hypoxic events during sleep, and is associated with substantial neurocognitive morbidity, particularly in children. Intermittent hypoxia (IH) leads to increases in apoptosis in the cortex and hippocampus of the adult rat, peaking at 48 h of exposure. To examine whether the susceptibility to IH exhibits developmental differences, rats were exposed to 48 h of IH at ages 2, 5, 10, 15, 20, 25, 30, 60, and 120-day postnatally, and apoptosis was determined by terminal deoxy-nucleotidyl transferase-mediated in situ end labeling and immunohistochemical staining for single-stranded DNA. Although IH induced apoptosis at all postnatal ages, smaller increases were apparent in 2 and 5-day old (P < 0.01 vs. any other age) while peak apoptosis occurred at 10-25 days (P < 0.001 vs. 30, 60, and 120 days). We conclude that a unique window of vulnerability to IH is present in the cortex and hippocampus during post-natal maturation, and may underlie the high frequency of neurobehavioral deficits associated with obstructive sleep apnea in children.
Subject(s)
Aging/physiology , Animals, Newborn/growth & development , Cerebral Cortex/physiology , Hippocampus/physiology , Hypoxia/etiology , Hypoxia/physiopathology , Animals , Apoptosis/physiology , Disease Susceptibility , Male , Rats , Rats, Sprague-Dawley , Reference ValuesABSTRACT
NF-kappaB, an ubiquitous transcription factor which plays a major role in the regulation of stress-related genes, is activated during environmental hypoxia in the dorsocaudal brainstem of adult rats. To examine the developmental pattern of NF-kappaB basal activity in the brainstem and the response to hypoxia, electromobility shift assays and immunohistochemical staining for the P65 subunit of NF-kappaB were performed in caudal brainstem samples of rats at 2, 5, 10, 15, and 60 days postnatal age, following normoxic or hypoxic (1 h in 10% O2) exposures. In addition, the expression of IkappaB-alpha, and IkappaB kinases (ikk)-alpha and -beta was also examined using Western blots. Basal NF-kappaB nuclear activity and nuclear P65 immunoreactivity increased with maturation. In contrast, hypoxia induced enhanced activation of NF-kappaB and nuclear translocation of P65 in youngest animals. Expression of both IkappaB-alpha and ikk-alpha was highest in the more immature rats, and decreased with postnatal age. In contrast, ikk-beta expression was unchanged over time. We conclude that NF-kappaB activity in caudal brainstem is developmentally regulated, and that hypoxia-induced NF-kappaB activation is more prominent in youngest rats. We postulate that postnatal regulation of NF-kappaB complex expression and function may underlie fundamental genomic processes mediating developmental changes in neuronal hypoxic tolerance.
Subject(s)
Brain Stem/growth & development , Brain Stem/metabolism , Hypoxia, Brain/metabolism , I-kappa B Proteins , NF-kappa B/metabolism , Acute Disease , Age Factors , Animals , Animals, Newborn , Brain Stem/chemistry , DNA-Binding Proteins/analysis , DNA-Binding Proteins/metabolism , Electrophoresis , Female , I-kappa B Kinase , Immunohistochemistry , NF-KappaB Inhibitor alpha , NF-kappa B/analysis , Pregnancy , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/metabolism , RatsABSTRACT
Intermittent hypoxia (IH) is the most frequent form of hypoxia occurring in the developing mammal. On one hand, the maturational process of neural, mechanical, pulmonary, and sleep state-dependent factors will favor the occurrence of IH during early postnatal life. On the other hand, it has also become clear that hypoxia, even when short lasting, can modify subsequent respiratory responses to hypoxia and induce a variety of genes whose consequences will persist for much longer periods than the duration of the hypoxic stimulus itself, i.e., functional and adaptive plasticities. The dynamic interactions between the overall duration and recurring frequency of IH, the severity of IH, and the level of neural maturity at the time of IH will modify the ventilatory, metabolic, and cardiovascular responses to hypoxia. We propose that the earlier IH will occur in the developmental course the more likely that the physiological responses to an ulterior hypoxic challenge will be altered even into adulthood. At this point in time, a critical examination of the field would suggest that the short-term alterations of the hypoxic ventilatory response (HVR) of the developing mammal to IH are qualitatively similar to those of the adult and display a biphasic pattern, namely, initial enhancement of the HVR followed by a reduction in HVR. However, the short- and long-term effects of IH on the modulation of neurotransmitter release, receptor binding and expression, intracellular signaling cascades, transcriptional regulation, and gene expression as a function of animal maturity are almost completely unknown. Further delineation of such complex responses to IH may permit the formulation of interventional strategies aiming at reducing the overall vulnerability of the young infant and child to apnea and sudden death.
Subject(s)
Gene Expression Regulation , Hypoxia/physiopathology , Respiratory Mechanics/physiology , Animals , Humans , Hypoxia/genetics , Mammals , Neuronal Plasticity , Signal Transduction/physiologyABSTRACT
Activation of platelet-derived growth factor receptor beta (PDGFR) within the caudal brainstem modulates the hypoxic ventilatory response. Since hypoxia does not induce apoptosis in the caudal brainstem, PDGFR could underlie such protective mechanism via a PI3 kinase-dependent phosphorylation of both Akt and BAD pathways. To further study this issue, caudal brainstem lysates were harvested from Sprague--Dawley rats during hypoxia (10% O(2)) after treatment with either vehicle or CGP 57148B (100 mg/kg), a selective blood-brain barrier-permeable PDGFR antagonist. Time-dependent increases in phosphorylated Akt occurred during hypoxia, peaking at 45' and lasting for up to 6 h, without parallel changes in total Akt protein. CGP 57148B attenuated Akt activation at all time points. Similarly, phosphorylation of BAD at serine136 but not at serine 112 occurred in the caudal brainstem as early as 15' of hypoxia, and was completely blocked by CGP 57148B. Furthermore, CGP 57148B treatment elicited significant increases in single-stranded DNA, caspase-like activity, and cleaved caspase 3 after 24 h of hypoxia that were absent in the caudal brainstem of hypoxic vehicle-treated animals. We conclude that PDGFR-dependent in vivo activation of both Akt and BAD during hypoxia prevents induction of apoptosis, and may contribute to the increased hypoxic tolerance of brainstem neurons.
Subject(s)
Apoptosis/drug effects , Carrier Proteins/drug effects , Hypoxia, Brain/metabolism , Nerve Degeneration/prevention & control , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/drug effects , Receptor, Platelet-Derived Growth Factor beta/drug effects , Solitary Nucleus/drug effects , Animals , Apoptosis/physiology , Benzamides , Carrier Proteins/metabolism , Caspases/drug effects , Caspases/metabolism , Cell Survival/drug effects , Cell Survival/physiology , DNA, Single-Stranded/metabolism , Enzyme Inhibitors/pharmacology , Hypoxia, Brain/pathology , Hypoxia, Brain/physiopathology , Imatinib Mesylate , Immunohistochemistry , Male , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Neurons/drug effects , Neurons/metabolism , Neuroprotective Agents/pharmacology , Phosphorylation/drug effects , Piperazines/pharmacology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, Platelet-Derived Growth Factor beta/metabolism , Respiratory Physiological Phenomena/drug effects , Signal Transduction/drug effects , Signal Transduction/physiology , Solitary Nucleus/metabolism , Solitary Nucleus/physiopathology , bcl-Associated Death ProteinABSTRACT
Murine exposure to silica is associated with enhanced tumor necrosis factor alpha (TNF-alpha) expression and matrix deposition. The regulation of TNF is mediated through TNF receptor (TNFR) activation of transcription factors. In the present work we have studied the importance of the individual TNFR in silica-induced lung inflammation and matrix deposition in mice. We studied RNA expression of TNF, alpha1(I) collagen, interstitial collagenase (MMP-13), and its inhibitor (TIMP-1) in the lungs of silica-treated mice. Furthermore, we correlated MMP-13/TIMP-1 RNA abundance with activation of the transcription factors AP-1 and NF-kappaB in the lungs of C57BL/6 mice, and of mice deficient in one of the two types of TNFR (p55(-/-) or p75(-/-)), exposed to silica (0.2 g/kg) or saline by intratracheal instillation. Animals were killed 28 d after exposure and lung hydroxyproline (HP), TNF, alpha1(I) collagen, MMP-13, and TIMP-1 RNA abundance was measured. AP-1 and NF-kappaB activation was studied by gel-shift assays. Compared with C57BL/6 mice, p55(-/-) and p75(-/-) mice significantly (*p < 0.05) decreased lung HP accumulation in response to silica. All murine strains enhanced TNF and alpha1(I) collagen mRNA in response to silica. Enhanced (p < 0.05) MMP-13 RNA expression was also observed in all murine strains in response to silica. Enhanced (p < 0.05) TIMP-1 RNA expression was observed in C57BL/6 mice, but not in p55(-/-) or p75(-/-) mice, in response to silica. NF-kappaB activation was observed in all murine strains, whereas AP-1 activation was observed only in C57BL/6 mice after silica treatment. These data suggest that TNFR deletion modifies MMP-13/ TIMP-1 expression in favor of matrix degradation.
Subject(s)
Collagenases/metabolism , Matrix Metalloproteinase 1/biosynthesis , Receptors, Tumor Necrosis Factor/deficiency , Silicosis/enzymology , Animals , Collagen/analysis , Female , Lung/chemistry , Matrix Metalloproteinase 13 , Mice , Mice, Inbred C57BLABSTRACT
Activation of platelet-derived growth factor-beta (PDGF-beta) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-beta receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O(2), and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-beta receptor expression. Although no significant changes in PDGF-beta receptor mRNA occurred over time, marked attenuation of PDGF-beta receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH (r: -0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB (n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia (n = 10). We conclude that decreased expression of PDGF-beta receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-beta receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.
Subject(s)
Adaptation, Physiological , Gene Expression , Hypoxia/physiopathology , Pulmonary Ventilation , Receptor, Platelet-Derived Growth Factor beta/genetics , Animals , Becaplermin , Blotting, Northern , Blotting, Western , Male , Microinjections , Oxygen/administration & dosage , Platelet-Derived Growth Factor/administration & dosage , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins c-sis , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptor, Platelet-Derived Growth Factor beta/physiology , Transcription, GeneticABSTRACT
The nucleus tractus solitarii (nTS) provides the initial central synaptic relay to peripheral chemoreceptor afferent inputs elicited by changes in oxygen tension. Insofar, the overall cumulative evidence pointing towards the N-methyl-D-aspartate (NMDA) glutamate receptor as the critical receptor underlying the early component of the hypoxic ventilatory response (HVR) is reviewed in detail. In addition, we will present recent findings supporting a role for platelet-derived growth factor (PDGF) beta receptor activation in modulation of the late phase of HVR. This evidence underscores the proposal of a working model for intracellular signaling pathways, downstream to the NMDA glutamate and PDGF-beta receptors in nTS neurons, which may contribute to both the ventilatory characteristics of the acute hypoxic response and to subsequently occurring functional adaptations and synaptic plasticity phenomena.
Subject(s)
Hypoxia/physiopathology , Respiratory Physiological Phenomena , Solitary Nucleus/physiopathology , Acute Disease , Animals , Gene Expression Regulation , Humans , Models, Biological , Nitric Oxide Synthase/metabolism , Protein Kinase C/metabolism , Receptors, N-Methyl-D-Aspartate/physiology , Receptors, Platelet-Derived Growth Factor/physiology , Signal TransductionABSTRACT
The early phase of the biphasic ventilatory response to hypoxia in mammals is critically dependent on NMDA glutamate receptor activation within the nucleus of the solitary tract. However, the mechanisms underlying the subsequent development of the typical ventilatory roll-off are unclear and could underlie important roles in the functional and molecular adaptation to oxygen deprivation. Because the growth factor platelet-derived growth factor (PDGF)-BB can modulate the open channel probability of NMDA receptors by activating PDGF-beta receptors, its contribution to hypoxic ventilatory roll-off was examined. Administration of PDGF-BB, but not PDGF-AA, in the nucleus of the solitary tract was associated with significant attenuations of the early hypoxic ventilatory response in conscious rats. Furthermore, marked reductions in the magnitude of hypoxic ventilatory roll-off occurred in mice heterozygous for a mutation in the PDGF-beta receptor. Administration of a PDGF-beta receptor antagonist to wild-type littermates elicited similar declines in hypoxic ventilatory roll-off. The relative abundance of PDGF-beta receptors was confirmed in the nucleus of the solitary tract and other nuclei implicated in the hypoxic ventilatory response. In nucleus of the solitary tract lysates, PDGF-beta receptor tyrosine phosphorylation was temporally correlated with hypoxic ventilatory roll-off formation. Increased PDGF-B chain mRNA expression was induced by hypoxia in the nucleus of the solitary tract, and PDGF-B chain immunoreactivity colocalized with approximately 40% of nucleus of the solitary tract neurons, demonstrating hypoxia-induced c-Fos enhancements. Thus, PDGF-BB release and PDGF-beta receptor activation in the nucleus of the solitary tract are critical components of hypoxic ventilatory roll-off and may have important functional implications in processes underlying survival and acclimatization to hypoxic environments.
Subject(s)
Brain Stem/metabolism , Hypoxia/physiopathology , Receptor, Platelet-Derived Growth Factor beta/physiology , Respiration , Animals , Becaplermin , Immunoblotting , Immunohistochemistry , In Situ Hybridization , Male , Mice/genetics , Mutation/physiology , Platelet-Derived Growth Factor/pharmacology , Precipitin Tests , Proto-Oncogene Proteins c-sis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Platelet-Derived Growth Factor beta/genetics , Solitary Nucleus/metabolism , Solitary Nucleus/physiologyABSTRACT
To study in vivo phosphorylation of N-methyl-D-aspartate (NMDA) glutamate receptors and the recruitment of protein kinase C isoforms during acute hypoxia, dorsocaudal brainstem lysates were harvested from conscious rats exposed to either room air or hypoxia (10% O2 for 5 and 15 min). Increased phosphorylation of the NR-1 subunit at serine residue 896 occurred during hypoxia and was blocked by pre-treatment with MK-801. Immunoblots of soluble and particulate fractions revealed subcellular translocation for PKC-beta, -gamma, -delta, -epsilon, and -iota during hypoxia with no changes in PKC-alpha, -mu, and -zeta. Translocation of PKC-beta, -delta and -epsilon was selectively attenuated following MK-801. We demonstrate that hypoxia leads to PKC-mediated activation of NMDA receptors in the brainstem, and that PKC-beta, -delta and -epsilon are the most likely candidates for NR-1 phosphorylation.
Subject(s)
Brain Stem/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Hypoxia/metabolism , Isoenzymes/physiology , Nerve Tissue Proteins/physiology , Protein Kinase C/physiology , Protein Processing, Post-Translational , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction , Animals , Biological Transport , Consciousness , Dizocilpine Maleate/pharmacology , Enzyme Activation , Phosphorylation , Rats , Rats, Sprague-DawleyABSTRACT
In the nucleus tractus solitarii, NMDA glutamate receptors are critical to the hypoxic ventilatory response. However, the signal transduction pathways underlying the hypoxic ventilatory response remain undefined. To assess the effect of a moderate hypoxic stimulus (10% O2) on tyrosine phosphorylation of proteins in the nucleus tractus solitarii, tissue lysates were harvested by repeated punch sampling at 0, 1, 10, and 60 min of hypoxia and examined for the presence of phosphorylated tyrosine residues by immunoblotting. Time-dependent phosphotyrosine increases occurred in proteins migrating at regions corresponding to molecular masses of 38-42, 50, 55, and 60 kDa, which were attenuated by pretreatment with the NMDA receptor channel blocker, MK-801. As extracellular signal-regulated kinase (Erk) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation may induce Fos and Jun gene transcription and activator protein-1 (AP-1) DNA binding, the activation of Erk1, Erk2, p38, and SAPK/JNK was examined in the nucleus tractus solitarii and neocortex during hypoxia and following administration of MK-801. Hypoxia enhanced Erk1, Erk2, and p38 activity in the cortex, but not in the nucleus tractus solitarii. Increased phosphorylation of SEK1 and SAPK/JNK-2 occurred in the nucleus tractus solitarii during hypoxia, whereas both SAPK/JNK-1 and SAPK/JNK-2 were recruited in cortex. MK-801 attenuated hypoxia-induced SEK1, SAPK/JNK-2, and AP-1 binding in the nucleus tractus solitarii, and the widespread activation of all MAP kinases in the cortex was also attenuated. We conclude that in conscious rats, a moderate hypoxic stimulus elicits NMDA-dependent widespread mitogen-activated protein kinase activation in cortex, but selective SAPK/JNK-2 and AP-1 activation in the nucleus tractus solitarii, thereby suggesting a functional role for the SAPK/JNK-2-AP-1 pathway.
Subject(s)
Hypoxia, Brain/physiopathology , Hypoxia/physiopathology , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinases , Protein Kinases/metabolism , Solitary Nucleus/enzymology , Adaptor Protein Complex alpha Subunits , Adaptor Proteins, Vesicular Transport , Animals , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Consciousness , DNA-Binding Proteins/physiology , Male , Membrane Proteins/analysis , Mitogen-Activated Protein Kinase 9 , Phosphorylation , Protein Binding/physiology , Protein Kinases/analysis , Rats , Rats, Sprague-Dawley , Respiration , Signal Transduction/physiology , Solitary Nucleus/chemistry , Transcription Factors/physiology , Tyrosine/metabolismABSTRACT
Nitric oxide (NO) is an excitatory neurotransmitter in the hypoxic ventilatory response (HVR). Furthermore, neuronal NO synthase (nNOS) activity in the developing rat correlates with the magnitude of late hypoxic ventilatory depression. To test the hypothesis that repeated short exposures to hypoxia may modify late HVR characteristics in young rats, we conducted 30-min hypoxic challenges in 2- to 3-day-old rat pups, before (Pre) and 6 h after (Post) they completed a series of eight cycles consisting of 5 min of hypoxia and 10 min of normoxia (Hyp-Norm) or normoxia throughout (Norm-Norm). In an additional group, similar challenges were performed after administration of either intraperitoneal vehicle or 25 mg/kg 7-nitroindazole (7-NI). Ventilation (VE) was measured using whole body plethysmography. Although no changes in peak VE responses occurred with episodic hypoxia (Pre vs. Post, P = not significant), late VE reductions were markedly attenuated in Post (DeltaVE from early to late: 7.2 +/- 1.5 ml/min in Pre vs. 4.5 +/- 1.1 ml/min in Post; P < 0.002). Furthermore, 7-NI treatment of Post animals was associated with late VE reductions to Pre levels in Hyp-Norm-exposed animals. Western blots of protein equivalents from the caudal brain stem revealed increased nNOS expression in Hyp-Norm compared with Norm-Norm (P < 0.01). Current findings suggest that repeated short hypoxic exposures improve the ability to sustain VE, which appears to be mediated by increased nNOS expression and activity in brain stem respiratory regions. We postulate that changes in nNOS may play a role in respiratory control plasticity.
Subject(s)
Aging/physiology , Animals, Newborn/physiology , Hypoxia/physiopathology , Nitric Oxide Synthase/physiology , Periodicity , Respiration , Animals , Animals, Newborn/growth & development , Brain Stem/enzymology , Immunoblotting , Nitric Oxide Synthase Type I , Rats , Rats, Sprague-DawleyABSTRACT
Stimulation of alveolar macrophages (AM) with adenosine-5-diphosphate (ADP) results in transient production of superoxide anion radical (O2.-; superoxide) and H2O2 in a metabolic event known as the respiratory burst. Initiation of the respiratory burst appears to depend on activation of protein kinase activity, whereas protein phosphatases might involved in termination of the burst. The involvement of protein kinase C was suggested by inhibition by bisindolylmaleimide I (GF 109203X), a relatively specific inhibitor. KN-62, an inhibitor of calcium-calmodulin protein kinase II, also partly inhibited the respiratory burst stimulated by ADP and phorbol esters. The role of protein phosphatases in termination of the ADP-stimulated respiratory burst of AM was examined with calyculin A (CA) (25-75 nM) or okadaic acid (OA) (1-5 microM), two inhibitors of protein phosphatase 1 and 2a (PP1;PP2a). A dose-dependent prolongation of the respiratory burst was observed in the presence of these inhibitors. CA and OA also markedly enhanced the rate of superoxide production stimulated by ADP, consistent with involvement of PP1/PP2a in regulating both the rate of activation and timing of termination. Treatment of AM with cyclosporin A (CsA) (1-50 microM), an inhibitor of the calcium-dependent protein phosphatase 2b (PP2b), stimulated superoxide production by itself and significantly prolonged the duration of ADP-stimulated superoxide production. CsA, however, did not increase the ADP-stimulated rate of superoxide production. Thus, PP1/PP2a appear to be the primary phosphatases for controlling the intensity of the respiratory burst during receptor-elicited superoxide production in AM, whereas PP1/PP2a and PP2b play a role in turning off the respiratory burst.
Subject(s)
Macrophages, Alveolar/metabolism , Proteins/metabolism , Superoxides/metabolism , Adenosine Diphosphate/pharmacology , Animals , In Vitro Techniques , Macrophages, Alveolar/drug effects , NADPH Oxidases/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Kinases/metabolism , Protein Phosphatase 1 , Rats , Rats, Sprague-Dawley , Respiratory Burst/drug effectsABSTRACT
Protein kinase C (PKC) activation in the nucleus tractus solitarii (NTS) is critical for mounting an appropriate hypoxic ventilatory response (HVR). Furthermore, hypoxia elicits translocation of both Ca2+-dependent and Ca2+-independent PKC isoforms in the NTS. However, the relative functional contribution of such PKC isoforms in mediating HVR is unclear. To study these issues, chronically instrumented adult Sprague-Dawley rats underwent hypoxic challenges (10% O2 balance in N2) following dorsocaudal brainstem microinjections of the selective Ca2+-dependent PKC inhibitor Gö 6976 (10 mmol in 1 microl). Compared with vehicle, Gö 6976 did not modify normoxic ventilation but maximally attenuated HVR by 38.4 +/- 6.7% (n = 9; P < 0.01), with similar contributions from tidal volume and respiratory frequency. In seven additional animals, when the non Ca2+-selective PKC blocker BIM I was concurrently microinjected with Gö 6976, further reductions in peak ventilatory responses to hypoxia occurred (P < 0.04). When BIM V, the inactive analog, was microinjected with Gö 6976, the magnitude of HVR attenuation was unchanged (n = 6; Gö 6976 vs. Gö 6976 + BIM V: P = NS). We conclude that in the dorsocaudal brainstem, PKC-mediated components of HVR involve activation of both Ca2+-dependent and Ca2+-independent PKC isoforms.
Subject(s)
Brain Stem/physiology , Calcium/physiology , Hypoxia/physiopathology , Protein Kinase C/physiology , Respiratory Mechanics/physiology , Animals , Blood Gas Analysis , Carbazoles/pharmacology , Enzyme Inhibitors/pharmacology , Hemodynamics/drug effects , Indoles/pharmacology , Male , Protein Kinase C/antagonists & inhibitors , Rats , Rats, Sprague-DawleyABSTRACT
In the nucleus of the solitary tract, NMDA receptors are critical for the hypoxic ventilatory response while neuronal nitric oxide synthase (NOS) modulates the late component of this response. Nuclear factor (NF)-kappaB is a ubiquitous transcription factor that increases the expression of multiple stress-activated genes. We sought to examine temporal changes in expression of NF-kappaB within the dorsocaudal brain stem of conscious rats after exposures to 10% O2. Time-dependent increases in NF-kappaB occurred with hypoxia and peaked at 60 min. Pretreatment with the N-methyl-D-aspartate (NMDA)-receptor channel antagonist dizocilpine maleate (MK-801) markedly attenuated NF-kappaB complexes during hypoxia. In contrast, after NOS inhibition with NG-nitro-L-arginine methyl ester (L-NAME), although NF-kappaB was diminished in normoxia, increased NF-kappaB expression still occurred with hypoxia. Increased phosphorylation of the NF-kappaB regulatory unit [inhibitory (I)kappaB] was detected by immunoblotting and also peaked at 60 min. Phosphorylation of Ikappa-B during hypoxia was attenuated by MK-801 but not by L-NAME. Thus NMDA-receptor activation in the dorsocaudal brain stem during hypoxia elicits in NF-kappaB activity marked enhancements that are unaffected after NOS blockade.
Subject(s)
Brain Stem/metabolism , Dizocilpine Maleate/pharmacology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Hypoxia/metabolism , NF-kappa B/biosynthesis , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Animals , Blotting, Western , Brain Stem/drug effects , NF-kappa B/drug effects , Phosphorylation , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
This study aimed to determine the role of protein kinase C (PKC) in signal transduction mechanisms underlying ventilatory regulation in the nucleus tractus solitarii (NTS). Microinjection of phorbol 12-myristate 13-acetate into the commissural NTS of nine chronically instrumented, unrestrained rats elicited significant cardiorespiratory enhancements that lasted for at least 4 h, whereas administration of vehicle (n = 15) or the inactive phorbol ester 4alpha-phorbol 12,13-didecanoate (n = 7) did not elicit minute ventilation (VE) changes. Peak hypoxic VE responses (10% O2-balance N2) were measured in 19 additional animals after NTS microinjection of bisindolylmaleimide (BIM) I, a selective PKC inhibitor (n = 12), BIM V (inactive analog; n = 7), or vehicle (Con; n = 19). In Con, VE increased from 139 +/- 9 to 285 +/- 26 ml/min in room air and hypoxia, respectively, and similar responses occurred after BIM V. BIM I did not affect room air VE but markedly attenuated hypoxia-induced VE increases (128 +/- 12 to 167 +/- 18 ml/min; P < 0. 02 vs. Con and BIM V). When BIM I was microinjected into the cerebellum (n = 4), cortex (n = 4), or spinal cord (n = 4), VE responses were similar to Con. Western blots of subcellular fractions of dorsocaudal brain stem lysates revealed translocation of PKCalpha, beta, gamma, delta, epsilon, and iota isoenzymes during acute hypoxia, and enhanced overall PKC activity was confirmed in the particulate fraction of dorsocaudal brain stem lysates harvested after acute hypoxia. These studies suggest that, in the adult rat, PKC activation in the NTS mediates essential components of the acute hypoxic ventilatory response.