Effects of increases in carboxyhemoglobin percent saturation and tissue hypoxia on carbon monoxide binding to canine skeletal and heart muscle extravascular tissue.

The major goal of this article was to quantify relationships of the carboxyhemoglobin % saturation, a calculated tissue PCO, and tissue hypoxia to the binding of carbon monoxide (CO) to canine skeletal and heart ventricular muscle extravascular (EV) tissue under normal conditions and during CO poisoning scenarios. These data are relevant to CO poisoning because CO bound to EV cellular hemoproteins evoke metabolic changes that produce toxic effects. Skeletal and heart muscle EV CO contents were calculated from data obtained from biopsies performed on living anesthetized dogs reported in previous publications (Coburn RF, Mayers LB. Am J Physiol 220: 66-74, 1971; Coburn RF, Ploegmakers F, Gondrie P, Abboud R. Am J Physiol 224: 870-876, 1973). Results include normal values of EV CO contents of resting skeletal muscle and heart ventricular muscle, effects of increasing COHb% saturation and a calculated mean tissue PCO on skeletal muscle EV CO binding, and effects of tissue hypoxia evoked by arterial hypoxemia on EV CO binding in both of these tissues. This study is the first that shows that tissue hypoxia-induced CO shifts out of blood resulting in increased EV CO binding are a mechanism that causes CO toxicity. Projections of results to tissue PCO levels occurring during different severe CO toxicity scenarios predict that skeletal muscle EV CO contents could increase as much as 100 to 300 fold.NEW & NEWSWORTHY This article provides quantification of some physiological parameters that determine carbon monoxide (CO) binding to skeletal and heart muscle extravascular tissue hemoproteins during normal and CO poisoning conditions. This is important information because toxicity occurring during CO poisoning is determined in part by CO binding to these proteins that results in detrimental changes in cellular metabolism.

Carbon Monoxide (CO), Nitric Oxide, and Hydrogen Sulfide Signaling During Acute CO Poisoning.

Major toxic effects of acute carbon monoxide (CO) poisoning result from increases in reactive oxygen species (ROS) and reactive nitrogen species (RNS) producing oxidative stress. The importance of altered nitric oxide (NO) signaling in evoking increases in RNS during CO poisoning has been established. Although there is extensive literature describing NO and hydrogen sulfide (H2S) signaling in different types of cells under normal conditions, how CO poisoning-evoked deregulation of additional NO signaling pathways and H2S signaling pathways could result in cell injury has not been previously considered in detail. The goal of this article was to do this. The approach was to use published data to describe signaling pathways driven by CO bonding to different ferroproteins and then to collate data that describe NO and H2S signaling pathways that could interact with CO signaling pathways and be important during CO poisoning. Arteriolar smooth muscle cells-endothelial cells located in the coronary and some cerebral circulations-were used as a model to illustrate major signaling pathways driven by CO bonding to different ferroproteins. The results were consistent with the concept that multiple deregulated and interacting NO and H2S signaling pathways can be involved in producing cell injury evoked during acute CO poisoning and that these pathways interact with CO signaling pathways.

Coronary and cerebral metabolism-blood flow coupling and pulmonary alveolar ventilation-blood flow coupling may be disabled during acute carbon monoxide poisoning.

Current evidence indicates that the toxicity of carbon monoxide (CO) poisoning results from increases in reactive oxygen species (ROS) generation plus tissue hypoxia resulting from decreases in capillary Po2 evoked by effects of increases in blood [carboxyhemoglobin] on the oxyhemoglobin dissociation curve. There has not been consideration of how increases in Pco could influence metabolism-blood flow coupling, a physiological mechanism that regulates the uniformity of tissue Po2, and alveolar ventilation-blood flow coupling, a mechanism that increases the efficiency of pulmonary O2 uptake. Using published data, I consider hypotheses that these coupling mechanisms, triggered by O2 and CO sensors located in arterial and arteriolar vessels in the coronary and cerebral circulations and in lung intralobar arteries, are disrupted during acute CO poisoning. These hypotheses are supported by calculations that show that the Pco in these vessels can reach levels during CO poisoning that would exert effects on signal transduction molecules involved in these coupling mechanisms.NEW & NOTEWORTHY This article introduces and supports a postulate that the tissue hypoxia component of carbon monoxide poisoning results in part from impairment of physiological adaptation mechanisms whereby tissues can match regional blood flow to O2 uptake, and the lung can match regional blood flow to alveolar ventilation.

The partial pressure of carbon monoxide in human tissues calculated using a parallel capillary-tissue cylinder model.

Tissue PCO values have not been previously estimated under conditions where the blood carboxyhemoglobin % saturation ([COHb]) is at a normal level or increased. Tissue PCO values are not known for conditions when [COHb] is increased during CO therapy or during CO poisoning. Using a modified Krogh parallel capillary-tissue model, mean tissue PCO was calculated for when [COHb] was 1, 5, 10, and 15% saturation, relevant to CO therapy, and 20, 30, and 40% saturation, relevant to CO poisoning. Calculations were made for the time during which CO was being inhaled, after cessation of CO uptake, and for different O2 extractions from blood flowing in the model capillary. The T1/2 of relevant CO reactions was used in these calculations. When the [COHb] increased to 5 to 10% saturation, mean tissue PCO values increased to 500 to 1,100% of values when the [COHb] was 1% saturation. When the [COHb] increased to 20 to 40% saturation, mean tissue PCO values increased to 2,300 to 5,700% of the 1% saturation value. Results indicate the utility of the modified Krogh model in furthering understanding the physiology of determinants of tissue PCO and should facilitate future studies of in vivo CO binding to different extravascular heme proteins during CO therapy and during CO poisoning. NEW & NOTEWORTHY Tissue PCO levels resulting from carboxyhemoglobin concentrations achieved during CO therapy or during CO poisoning have not been previously estimated. Results published here show that at carboxyhemoglobin levels achieved during CO therapy there are 500 to 1,100% increases in mean tissue PCO values. With carboxyhemoglobin increases associated with toxic effects, there are 2,300 to 5,700% increases in the mean tissue PCO. These differences suggest a basis for understanding the therapeutic and toxic effects of CO.

A possible carbon monoxide shuttle in the lung.

The Coburn, Forster, Kane Equation (CFKE) describes a current understanding of the physiology of lung uptake and excretion of carbon monoxide (CO). The lung mean capillary PCO is an important term in this equation because it drives CO excretion and functions as "back-pressure" during uptake of exogenous CO. Results of previous studies have indicated that the mean capillary PCO of normal human lungs is equal to values calculated using the Haldane Equation, as described by the CFKE. The physiological explanation of how this parameter is set at this level is unknown. As a possible explanation, this study tested a hypothesis that a CO shuttle could be involved. Results of calculation-simulations indicate that a CO shuttle operates in a single alveolus model and imply that it could function as a determinant of the lung mean capillary PCO.

Carbon monoxide uptake and excretion: testing assumptions made in deriving the Coburn-Forster-Kane equation.

The Coburn-Forster-Kane equation (CFKE) describes physiological variables that define pulmonary carbon monoxide (CO) uptake and excretion, and carboxyhemoglobin (COHb) levels. This equation is useful in predicting CO uptakes and COHb values in normal humans. However, some assumptions made in its derivation have never been proven and whether or not it accurately describes the physiology of CO excretion is not known. Two assumptions specifically addressed are: that the mean capillary PCO can be computed using the Haldane equation; and that the diffusing capacity relevant to CO uptake is equal to the diffusing capacity relevant to CO excretion. Both assumptions were supported by results obtained in this study. These findings plus results of experiments that compared measured and CFKE-calculated changes in the body CO stores suggest that the CFKE accurately describes the physiology of both pulmonary CO excretion and uptake.

The measurement of endogenous carbon monoxide production.

Recent findings that heme oxygenase-1 can be induced by oxidative stress and inflammation in many different cellular systems, and that carbon monoxide (CO) produced as a by-product of this enzyme is a signaling molecule, have generated a major research area with hundreds of studies published over the last few years. The measurement of expired CO concentration has been used in humans as a biomarker of induced heme oxygenase resulting from inflammation or oxidative stress, but a precise method of measuring endogenous CO production that can be easily used to study patients is needed. The present study describes such a method. The described method allows calculation of the rate of heme catabolism with a precision of ±2 µmol/h, ∼10% of the mean normal rate in subjects used in this investigation. This method, which is subject-patient friendly, precise, and inexpensive to perform, should be applicable to studies performed on humans with induced heme oxygenase and studies of effects of therapy for inflammatory and hemolytic diseases.

Polyamine effects on cell function: Possible central role of plasma membrane PI(4,5)P2.

The polyamines spermidine, spermine, and putrescine are intimately involved in and required for cell growth and proliferation. There are also multiple effects of polyamines on other cellular processes that seem not to be a result of changes in protein expression. It is a daunting task to classify and understand cellular effects of endogenous polyamines. There has been no central hypothesis how these effects can occur or how spermine and spermidine could be targeted to various signal transduction cascades. However, now there is evidence that multiple effects of endogenous polyamines on different cellular processes may involve plasma membrane PI(4,5)P(2) and recent evidence of how polyamines could be targeted to specific cellular functions.

Polyamines, PI(4,5)P2, and actin polymerization.

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.

Smooth muscle raft-like membranes.

We developed a method for extracting raft-like, liquid-ordered membranes from the particulate fraction prepared from porcine trachealis smooth muscle. This fraction, which contains most of the plasma membrane in this tissue, was homogenized in the presence of cold 0.5% Triton X-100. After centrifugation, membranes containing high contents of sphingomyelin (SM) and cholesterol and low phosphatidylcholine (PC) contents remained in the pellet. Thirty-five millimolar octyl glucoside (OG) extracted 75% of these membranes from the Triton X-100-resistant pellet. These membranes had low buoyant densities and accounted for 28% of the particulate fraction lipid. Their lipid composition, 22% SM, 60% cholesterol, 11% phosphatidylethanolamine, 8% PC, <1% phosphatidylinositol, and coisolation with 5'-nucleotidase and caveolin-1 suggest that they are liquid-ordered membranes. We compared characteristics of OG and Triton X-100 extractions of the particulate fraction. In contrast to Triton X-100 extractions, membranes released from the particulate fraction by OG were mainly collected in low buoyant fractions at densities ranging from 1.05 to 1.11 g/ml and had phospholipid and cholesterol contents consistent with a mixture of liquid-ordered and liquid-disordered membranes. Thus, OG extraction of apparent liquid-ordered membranes from Triton X-100-resistant pellets was not due to selective extraction of these membranes. Low buoyant density appears not to be unique for liquid-ordered membranes.

Spermine increases phosphatidylinositol 4,5-bisphosphate content in permeabilized and nonpermeabilized HL60 cells.

The polyamine spermine (N,N'bis[3-aminopropyl]-1,4-butanediamine) activates phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P5K) and phosphatidylinositol 4-kinase (PtdIns4K) in vitro. Spermine concentration increases that occur in proliferating cells were approximated in streptolysin O-permeabilized HL60 cells. When phospholipase C was activated by GTPgammaS in the presence of PITPalpha, 0.1-1.2 mM spermine evoked increases in PtdIns(4,5)P(2) contents in a dose-dependent manner to 110-170% of control and concomitantly decreased inositol phosphate formation by 10-50%. Spermine-induced increases in PtdIns(4,5)P(2) content in permeabilized cells also occurred during GTPgammaS stimulation in the absence of PITPalpha, were augmented in the presence of PITPalpha, occurred in unstimulated cells and were additive to PtdIns(4,5)P(2) formation evoked by ARF1, another activator of phosphoinositide kinases. Slowly developing spermine-evoked increases in PtdIns(4,5)P(2) contents occurred in nonpermeabilized cells that were abolished in the presence of a spermine transport inhibitor. Data are consistent with spermine at physiological concentrations evoking a PITPalpha-dependent shift in formation of PtdIns(4,5)P(2) from compartments that contained an active phospholipase C to compartments that were separated from an active PLC and from PtdIns(4,5)P(2) formed by ARF1.