Barry W. Allen

Hemoglobin, nitric oxide and molecular mechanisms of hypoxic vasodilation

The protected transport of nitric oxide (NO) by hemoglobin (Hb) links the metabolic activity of working tissue to the regulation of its local blood supply through hypoxic vasodilation. This physiologic mechanism is allosterically coupled to the O(2) saturation of Hb and involves the covalent binding of NO to a cysteine residue in the beta-chain of Hb (Cys beta93) to form S-nitrosohemoglobin (SNO-Hb). Subsequent S-transnitrosation, the transfer of NO groups to thiols on the RBC membrane and then in the plasma, preserves NO vasodilator activity for delivery to the vascular endothelium. This SNO-Hb paradigm provides insight into the respiratory cycle and a new therapeutic focus for diseases involving abnormal microcirculatory perfusion. In addition, the formation of S-nitrosothiols in other proteins may regulate an array of physiological functions.

Two faces of nitric oxide: implications for cellular mechanisms of oxygen toxicity

Recent investigations have elucidated some of the diverse roles played by reactive oxygen and nitrogen species in events that lead to oxygen toxicity and defend against it. The focus of this review is on toxic and protective mechanisms in hyperoxia that have been investigated in our laboratories, with an emphasis on interactions of nitric oxide (NO) with other endogenous chemical species and with different physiological systems. It is now emerging from these studies that the anatomical localization of NO release, which depends, in part, on whether the oxygen exposure is normobaric or hyperbaric, strongly influences whether toxicity emerges and what form it takes, for example, acute lung injury, central nervous system excitation, or both. Spatial effects also contribute to differences in the susceptibility of different cells in organs at risk from hyperoxia, especially in the brain and lungs. As additional nodes are identified in this interactive network of toxic and protective responses, future advances may open up the possibility of novel pharmacological interventions to extend both the time and partial pressures of oxygen exposures that can be safely tolerated. The implications of a better understanding of the mechanisms by which NO contributes to central nervous system oxygen toxicity may include new insights into the pathogenesis of seizures of diverse etiologies. Likewise, improved knowledge of NO-based mechanisms of pulmonary oxygen toxicity may enhance our understanding of other types of lung injury associated with oxidative or nitrosative stress.

Cerebral blood flow and brain oxygenation in rats breathing oxygen under pressure.

Hyperbaric oxygen (HBO2) increases oxygen tension (PO2) in blood but reduces blood flow by means of O2-induced vasoconstriction. Here we report the first quantitative evaluation of these opposing effects on tissue PO2 in brain, using anesthetized rats exposed to HBO2 at 2 to 6 atmospheres absolute (ATA). We assessed the contribution of regional cerebral blood flow (rCBF) to brain PO2 as inspired PO2 (PiO2) exceeds 1 ATA. We measured rCBF and local PO2 simultaneously in striatum using collocated platinum electrodes. Cerebral blood flow was computed from H2 clearance curves in vivo and PO2 from electrodes calibrated in vitro, before and after insertion. Arterial PCO2 was controlled, and body temperature, blood pressure, and EEG were monitored. Scatter plots of rCBF versus pO2 were nonlinear (R2 = 0.75) for rats breathing room air but nearly linear (R2 = 0.88–0.91) for O2 at 2 to 6 ATA. The contribution of rCBF to brain PO2 was estimated at constant inspired PO2, by increasing rCBF with acetazolamide (AZA) or decreasing it with N-nitro-l-arginine methyl ester (l-NAME). At basal rCBF (78 mL/100 g min), local PO2 increased 7- to 33-fold at 2 to 6 ATA, compared with room air. A doubling of rCBF increased striatal PO2 not quite two-fold in rats breathing room air but 13- to 64-fold in those breathing HBO2 at 2 to 6 ATA. These findings support our hypothesis that HBO2 increases PO2 in brain in direct proportion to rCBF.

Contributions of nitric oxide synthase isoforms to pulmonary oxygen toxicity, local vs. mediated effects

Reactive species of oxygen and nitrogen have been collectively implicated in pulmonary oxygen toxicity, but the contributions of specific molecules are unknown. Therefore, we assessed the roles of several reactive species, particularly nitric oxide, in pulmonary injury by exposing wild-type mice and seven groups of genetically altered mice to >98% O2 at 1, 3, or 4 atmospheres absolute. Genetically altered animals included knockouts lacking either neuronal nitric oxide synthase (nNOS(-/-)), endothelial nitric oxide synthase (eNOS(-/-)), inducible nitric oxide synthase (iNOS(-/-)), extracellular superoxide dismutase (SOD3(-/-)), or glutathione peroxidase 1 (GPx1(-/-)), as well as two transgenic variants (S1179A and S1179D) having altered eNOS activities. We confirmed our earlier finding that normobaric hyperoxia (NBO2) and hyperbaric hyperoxia (HBO2) result in at least two distinct but overlapping patterns of pulmonary injury. Our new findings are that the role of nitric oxide in the pulmonary pathophysiology of hyperoxia depends both on the specific NOS isozyme that is its source and on the level of hyperoxia. Thus, iNOS predominates in the etiology of lung injury in NBO2, and SOD3 provides an important defense. But in HBO2, nNOS is a major contributor to pulmonary injury, whereas eNOS is protective. In addition, we demonstrated that nitric oxide derived from nNOS is involved in a neurogenic mechanism of HBO2-induced lung injury that is linked to central nervous system oxygen toxicity through adrenergic/cholinergic pathways.

Electrochemical activation of electrodes for amperometric detection of nitric oxide

An open question in the literature of nitric oxide detection was investigated: does electrochemical activation account for the enhanced properties of certain presumed chemically-modified electrodes? Uniform electrodes of graphite, iridium, palladium, platinum, and ruthenium were exposed to potential cycling and then tested for amperometric response to nitric oxide to identify principles that govern electrochemical activation of nitric oxide electrodes. These electrodes were compared to similar electrodes that were not cycled. Only cycled graphite and ruthenium showed significantly increased responses. Graphite demonstrated enhanced performance after exposure to cycling potentials at which oxygen, CO(2), and soluble carbonates form, suggesting that erosion of the electrode enhanced its response by increasing the surface area accessible to nitric oxide. This may explain the performance of carbon fibers cycled to the same potentials in solutions containing metalloporphyrins. The response of ruthenium was enhanced after cycling to less extreme potentials at which soluble species do not form and at which a metallic conductive oxide, RuO(2), could lay down a stable, adherent layer on the electrode surface. Cycled ruthenium also exhibited a much greater increase in capacitance after cycling, consistent with the formation of a conductive surface layer.

Nitric oxide-mediated central sympathetic excitation promotes CNS and pulmonary O2 toxicity

In hyperbaric oxygen (HBO(2)) at or above 3 atmospheres absolute (ATA), autonomic pathways link central nervous system (CNS) oxygen toxicity to pulmonary damage, possibly through a paradoxical and poorly characterized relationship between central nitric oxide production and sympathetic outflow. To investigate this possibility, we assessed sympathetic discharges, catecholamine release, cardiopulmonary hemodynamics, and lung damage in rats exposed to oxygen at 5 or 6 ATA. Before HBO(2) exposure, either a selective inhibitor of neuronal nitric oxide synthase (NOS) or a nonselective NOS inhibitor was injected directly into the cerebral ventricles to minimize effects on the lung, heart, and peripheral circulation. Experiments were performed on both anesthetized and conscious rats to differentiate responses to HBO(2) from the effects of anesthesia. EEG spikes, markers of CNS toxicity in anesthetized animals, were approximately four times as likely to develop in control rats than in animals with central NOS inhibition. In inhibitor-treated animals, autonomic discharges, cardiovascular pressures, catecholamine release, and cerebral blood flow all remained below baseline throughout exposure to HBO(2). In control animals, however, initial declines in these parameters were followed by significant increases above their baselines. In awake animals, central NOS inhibition significantly decreased the incidence of clonic-tonic convulsions or delayed their onset, compared with controls. The novel findings of this study are that NO produced by nNOS in the periventricular regions of the brain plays a critical role in the events leading to both CNS toxicity in HBO(2) and to the associated sympathetic hyperactivation involved in pulmonary injury.

Autonomic activation links CNS oxygen toxicity to acute cardiogenic pulmonary injury.

Breathing hyperbaric oxygen (HBO₂), particularly at pressures above 3 atmospheres absolute, can cause acute pulmonary injury that is more severe if signs of central nervous system toxicity occur. This is consistent with the activation of an autonomic link between the brain and the lung, leading to acute pulmonary oxygen toxicity. This pulmonary damage is characterized by leakage of fluid, protein, and red blood cells into the alveoli, compatible with hydrostatic injury due to pulmonary hypertension, left atrial hypertension, or both. Until now, however, central hemodynamic parameters and autonomic activity have not been studied concurrently in HBO₂, so any hypothetical connections between the two have remained untested. Therefore, we performed experiments using rats in which cerebral blood flow, electroencephalographic activity, cardiopulmonary hemodynamics, and autonomic traffic were measured in HBO₂ at 5 and 6 atmospheres absolute. In some animals, autonomic pathways were disrupted pharmacologically or surgically. Our findings indicate that pulmonary damage in HBO₂ is caused by an abrupt and significant increase in pulmonary vascular pressure, sufficient to produce barotrauma in capillaries. Specifically, extreme HBO₂ exposures produce massive sympathetic outflow from the central nervous system that depresses left ventricular function, resulting in acute left atrial and pulmonary hypertension. We attribute these effects on the heart and on the pulmonary vasculature to HBO₂-mediated central sympathetic excitation and catecholamine release that disturbs the normal equilibrium between excitatory and inhibitory activity in the autonomic nervous system.

Baroreflex-mediated cardiovascular responses to hyperbaric oxygen.

The cardiovascular system responds to hyperbaric hyperoxia (HBO2) with vasoconstriction, hypertension, bradycardia, and reduced cardiac output (CO). We tested the hypothesis that these responses are linked by a common mechanism-activation of the arterial baroreflex. Baroreflex function in HBO2 was assessed in anesthetized and conscious rats after deafferentation of aortic or carotid baroreceptors or both. Cardiovascular and autonomic responses to HBO2 in these animals were compared with those in intact animals at 2.5 ATA for conscious rats and at 3 ATA for anesthetized rats. During O2 compression, hypertension was greater after aortic or carotid baroreceptor deafferentation and was significantly more severe if these procedures were combined. Similarly, the hyperoxic bradycardia observed in intact animals was diminished after aortic or carotid baroreceptor deafferentation and replaced by a slight tachycardia after complete baroreceptor deafferentation. We found that hypertension, bradycardia, and reduced CO--the initial cardiovascular responses to moderate levels of HBO2--are coordinated through a baroreflex-mediated mechanism initiated by HBO2-induced vasoconstriction. Furthermore, we have shown that baroreceptor activation in HBO2 inhibits sympathetic outflow and can partially reverse an O2-dependent increase in arterial pressure.

Electrochemical detection of nitric oxide in biological fluids

The challenges that must be overcome in order to detect nitric oxide (NO) in biological fluids include its low physiological concentration (1-nM) and its short half-life (a few seconds or less). Electrochemistry is capable of making such measurements, if certain principles, both biological and electrochemical, are kept in mind. We discuss these principles and demonstrate an example of practical measurement by detecting NO release in a drop of blood suspended within the reference electrode of an electrochemical cell. We elicit the NO release by decreasing the oxygen concentration in the gaseous atmosphere surrounding the drop.

Electrochemical detection of physiological nitric oxide: Materials and methods

Advances in the electroanalytical technology of NO detection make it possible to detect the release of robust concentrations of NO from living systems under pathological or pharmacological conditions. However, technical improvements should enable the construction of research instruments with one or two orders of magnitude improvement in both detection limit and temporal resolution. Such instruments would be capable of revealing physiological NO production and could help quantify the correlations between NO levels and health or disease, ultimately leading to important applications in biomedical research and clinical medicine.