Significant Increase In pO Research Articles

Human Erythrocyte Mechano-Activated K+ Channel A (HEMKCA): Effect of Applied Pressure on Open Probability, a Kinetic Study

Human erythrocyte (HE) has a life span of 120 days. Senescence of these cells has been linked to the cyclical stress faced during its circulation, especially to the mechanical stress suffered passing through capillaries.In our laboratory we studied the molecular processes underlying this biological clock, and had proposed a channel whose open probability (Po) shows a sigmoid dependence on the applied pressure: The Human Erythrocyte Machano-activated K+ Channel A (HEMKCA), which shows a PK/PNa of 100, a mean-conductance of aprox. 20 pS at zero current and is Ca2+ modulated (140mMKCl, 10mMNaCl, 1mMCaCl2, pH7.0)(1,2). We had proposed a new hypothesis for the senescent process of HE, with this channel playing a crucial roll as the molecular pressure sensor underlying its biological clock.Here using the patch clamp technique, HEMKCA activity was recorded and a kinetic analysis of the burst activity was performed. After obtaining a consistent model describing burst activity with two open and one close states (C1-O-C2)(3), the activity was recorded 100s before and after applying negative pressure in the patch pipette. Our results showed a significant increase in Po (n=9, p<0.05) and an increase in short-closed-state area (C2) of 0.833±0.057 to 0.909±0.055(n=7, p<0.05); this coupled with the fact that the τ obtained for the three proposed states did not undergo significant changes. The analysis of the rate constants shows that the increase in the proportion of short-closed-state (C2) was due to a decrease in the output frequency from open-state to the long-closed-state, K12 from 8,825±3,65 to 4,148±2,458seg−1(p<0.05).We conclude that the pressure does not exert its effect on the burst activity but rather decreases the probability of leaving this mode of activity.(1)(2005) Biophys. J.88(1):593.(2)(2008) Biophys. J.91(1):1101.(3)(2015) Biophys. J.108(2):439-440.

Comparison of Arterial and Venous Blood Gases and the Effects of Analysis Delay and Air Contamination on Arterial Samples in Patients with Chronic Obstructive Pulmonary Disease and Healthy Controls

Background: Arterial blood gases (ABGs) are often sampled incorrectly, leading to a ‘mixed’ or venous sample. Delays in analysis and air contamination are common. Objectives: We measured the effects of these errors in patients with chronic obstructive pulmonary disease (COPD) exacerbations and controls. Methods: Arterial and venous samples were analyzed from 30 patients with COPD exacerbation and 30 controls. Venous samples were analysed immediately and arterial samples separated into non-air-contaminated and air-contaminated specimens and analysed at 0, 30, 60, 90 and 180 min. Results: Mean venous pH was 7.371 and arterial pH was 7.407 (p < 0.0001). There was a correlation between venous and arterial pH (r = 0.5347, p < 0.0001). The regression equation to predict arterial pH was: arterial pH = 4.2289 + 0.43113 · venous pH. There were no clinically significant differences in arterial PO₂ associated with analysis delay. A statistically significant decline in pH was detected at 30 min in patients with COPD exacerbation (p = 0.0042) and 90 min in controls (p < 0.0001). A clinically significant decline in pH emerged at 73 min in patients with COPD exacerbation and 87 min in controls. Air contamination was associated with a clinically significant increase in PO₂ in all samples, including those that were immediately analyzed. Conclusions: Arterial and venous pH differ significantly. Venous pH cannot accurately replace arterial pH. Temporal delays in ABG analysis result in a significant decline in measured pH. ABGs should be analysed within 30 min. Air contamination leads to an immediate increase in measured PO₂, indicating that air-contaminated ABGs should be discarded.

Phosphorylation Activates RyR2 By Uncoupling The Channel From The Influence Of Cytosolic Ca2+ But Also Inhibits RyR2 By A Distinct Mechanism

Phosphorylation of RyR2 is generally thought to be associated with an increase in open probability (Po), however, there is disagreement over which phosphorylation sites are responsible. To investigate the gating mechanisms underlying phosphorylation-induced changes in RyR2 Po, we incorporated native sheep RyR2 into bilayers under voltage-clamp conditions with luminal Ca2+ as the permeant ion. Phosphorylation of RyR2 channels above basal levels, either by PKA or by activation of an endogenous kinase (by incubation with Mg2+ATP) leads to massive increases in Po that are associated with very long open states. The channels become effectively uncoupled from the influence of cytosolic Ca2+ because reductions in cytosolic [Ca2+] to sub-activating levels do not reduce open lifetimes nor significantly reduce Po. Treatment of channels with the phosphatase, PP1 reverses these gating changes returning the channels to a cytosolic Ca2+-sensitive mode of gating. In contrast, to the gating changes described above which are specific for phosphorylation of RyR2 above basal levels, complete dephosphorylation of RyR2 by PP1 treatment causes significant increases in Po. Po increased from 0.074±0.032 to 0.218±0.028 (SEM;n=4) after incubation with PP1 (5 units). The channel gating associated with completely dephosphorylated channels, or channels phosphorylated to 75% of maximum at serine-2809, demonstrates that the channels are sensitive to cytosolic [Ca2+]. We cannot yet assign specific phosphorylation sites to particular change in channel gating but it is clear that phosphorylation of RyR2 exerts effects which span the entire range of the RyR2 activity landscape. The huge scope for changing RyR2 channel activity suggests that phosphorylation of RyR2 is important for physiological regulation of the channel and that dysregulation of SR Ca2+-release would be expected with prolonged hyperphosphorylation.Supported by the BHF

Effects of Broiler Rearing Environment on Transmission of F-Strain Mycoplasma gallisepticum from Commercial Layer Hens to Broiler Chickens: Role of Acid-Base Balance

Two trials were conducted concurrently to determine and compare, blood pH, blood gases, hematocrit and hemoglobin in F-strain Mycoplasma gallisepticum (FMG) inoculated layers and FMG contactinfected broilers. At the termination of the study, FMG-inoculated layers had the highest partial pressure of O and the lowest partial pressure of CO as compared with the other treatment groups. Blood pH values 2 2 were unaffected by FMG inoculation. Hematocrit and blood concentrations of hemoglobin were slightly higher and HCO levels were lowest in FMG contact-infected broilers in comparison to the other treatments groups. 3 Mycoplasma gallisepticum inoculated layers also resulted in a significant increase in blood concentrations of K , a decrease in Na , but no significant effects on blood concentrations of Ca and Cl . There were no + + 2+ differences in plasma glucose, cholesterone, triglyceride and anion gap, but osmolality was significantly reduced in FMG contact-infected broilers. Results indicate that inoculation of layers with FMG vaccine results in changes in plasma acid-base status along with changes in other blood metabolic variables. However, the FMG inoculation did not prevent homeostatic regulation of acid-base balance, as indicated by constant blood pH. The significant increase in pO in FMG inoculated layers is generally associated with an oxygen2 dependent improvement in tissue oxygenation. Elevated arterial partial pressure of oxygen is beneficial to maximize oxygen transport capacity along with high concentrations of hemoglobin and hematocrit to carry oxygen throughout the body. It was concluded that in addition to protecting birds from MG infection, an FMG vaccine may improve the layer chicken’s ability to withstand the harmful effects of stressors on their performance and well-being.

Tissue pO 2 of Orthotopic 9L and C6 Gliomas and Tumor-Specific Response to Radiotherapy and Hyperoxygenation

Tumor hypoxia is a well-known therapeutic problem; however, a lack of methods for repeated measurements of glioma partial pressure of oxygen (pO(2)) limits the ability to optimize the therapeutic approaches. We report the effects of 9.3 Gy of radiation and carbogen inhalation on orthotopic 9L and C6 gliomas and on the contralateral brain pO(2) in rats using a new and potentially widely useful method, multisite in vivo electron paramagnetic resonance oximetry. Intracerebral 9L and C6 tumors were established in the left hemisphere of syngeneic rats, and electron paramagnetic resonance oximetry was successfully used for repeated tissue pO(2) measurements after 9.3 Gy of radiation and during carbogen breathing for 5 consecutive days. Intracerebral 9L gliomas had a pO(2) of 30-32 mm Hg and C6 gliomas were relatively hypoxic, with a pO(2) of 12-14 mm Hg (p < 0.05). The tissue pO(2) of the contralateral brain was 40-45 mm Hg in rats with either 9L or C6 gliomas. Irradiation resulted in a significant increase in pO(2) of the 9L gliomas only. A significant increase in the pO(2) of the 9L and C6 gliomas was observed in rats breathing carbogen, but this effect decreased during 5 days of repeated experiments in the 9L gliomas. These results highlight the tumor-specific effect of radiation (9.3.Gy) on tissue pO(2) and the different responses to carbogen inhalation. The ability of electron paramagnetic resonance oximetry to provide direct repeated measurements of tissue pO(2) could have a vital role in understanding the dynamics of hypoxia during therapy that could then be optimized by scheduling doses at times of improved tumor oxygenation.

Blockade of receptor for advanced glycation end product attenuates pulmonary reperfusion injury in mice

The receptor for advanced glycation end products (RAGE) is expressed at high levels in the lung, particularly in type 1 alveolar cells, and has been shown to amplify injury triggered by acute stress. Previous studies suggest serum concentrations of soluble RAGE increase during pulmonary reperfusion injury after transplantation. RAGE blockade has been shown to suppress hepatic and cardiac ischemia and reperfusion injury in mice. Thus we tested the hypothesis that RAGE mediates tissue-injury mechanisms in ischemia and reperfusion injury in the lung. C57BL/6 mice were subjected to 30 minutes of pulmonary ischemia by clamping the left hilum, followed by 60 minutes of reperfusion. Lung function was assessed by means of blood gas analysis, and capillary leak was assessed by injecting fluorescein isothiocyanate-labeled albumin and comparing fluorescence in bronchial lavage fluid with that in serum. Histologic analysis of the lung was performed by a pathologist naive to the experimental conditions. In animals subjected to RAGE blockade, significant increases in Po(2) (108 vs 73 mm Hg, P = .0094) and more than 3-fold decrease in capillary leak Relative Fluorescent Units (RFU, 6.12 vs 1.75; P = .001) were observed. Histologic examination revealed significant injury reduction in soluble RAGE-treated animals versus control animals. RAGE knockout mice exhibited a protected phenotype when exposed to pulmonary ischemia and reperfusion. Additionally, interleukin 8 production and nuclear factor kappaB activation were increased in control mice. Abrogation of RAGE signaling attenuates pulmonary ischemia and reperfusion injury. This study suggests that RAGE might play a central role in pulmonary reperfusion injury and in transplantation and that blockade of RAGE might offer a potential target to abrogate pulmonary reperfusion injury in clinical transplantation.

Potentiation of cyclophosphamide chemotherapy using the anti-angiogenic drug thalidomide: Importance of optimal scheduling to exploit the ‘normalization’ window of the tumor vasculature

The aim of this work was to study how administration schedule affects potentiation of cyclophosphamide, an alkylating agent, by thalidomide, an anti-angiogenic agent. Tumor oxygenation after thalidomide administration was determined over time by EPR oximetry. Such measurements provide a surrogate marker for determining the timing of ‘normalization’ of tumor vasculature. Re-growth delays were measured using different combinations and schedules of treatments. Additionally, the uptake of the metabolite of cyclophosphamide (hydroxycyclophosphamide or OH-CP) into tumors was determined by high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). A significant increase in pO 2 was observed after 2 and 3 days of treatment before eventually declining on day 4. Thalidomide potentiated the effect of cyclophosphamide only when cyclophosphamide was administered after 2 days of treatment with thalidomide (no significant benefit using other schedules). In this time frame, the HPLC/MS/MS measurements showed that the quantity of OH-CP penetrating into the tumor was about twice in mice treated by thalidomide compared to controls. In conclusion, the present study demonstrates that the benefit of combined therapy using an anti-angiogenic agent with a cytotoxic agent requires knowledge of the time window during which the vessels initially become normalized.

Continuous measurements of cerebral tissue oxygen pressure during hyperbaric oxygenation—HBO effects on brain edema and necrosis after severe brain trauma in rabbits

Introduction: Severe brain injury is one of the most frequent causes of severe disability in the young. In acute management of brain trauma, new approaches based on experimental animal investigations should be sought. Methods: Twenty male, juvenile Chinchilla-Bastard rabbits received standardized cold-injury-induced-brain-trauma (CIBT). A metal probe (temperature −196 °C) was applied epidurally over 10 s. The hyperbaric oxygenation (HBO) group ( n=10) underwent 90-min HBO sessions with 100% oxygen at 2.5 atmospheres absolute (1 h, 24±2 h, 48±2 h after CIBT). Cerebral tissue pO 2-measurements were performed 60 min after CIBT, during the three HBO sessions and on day 4. The control group ( n=10) underwent no treatment. Animals were sacrificed on day 4, and brains were analyzed histologically. Results: In the HBO group, pO 2 measurements showed a significant increase in pO 2 between day 1 and day 4, whereas no significant changes were observed in the control group. During the first HBO session, mean pO 2 was 169 mm Hg, during the second 305 mm Hg and during the third 420 mm Hg. The mean area of necrosis was 16.2 mm 2 in the HBO group, in the control group 19.9 mm 2. The areas of brain edema were significantly smaller in the HBO group. Mortality in the HBO group was 0%, in the control group 20%. Conclusion: HBO appears to be beneficial as an adjunct treatment of severe head trauma. To find optimal treatment protocols, further clinical studies must be developed.

NEUTROPHILS ARE ASSOCIATED WITH A FASTER RECOVERY AFTER MUSCLE INJURY

One of the first observed events after skeletal muscle injury is acute inflammation, which is thought to affect functional recovery. PURPOSE To determine whether neutrophils (PMN) are correlated with functional recovery after muscle injury. METHODS Male Sprague-Dawley rats (n=47, 314±60g) were subjected to an eccentric injury of the tibialis anterior (TA) by stimulating the peroneal nerve while plantar flexing the ankle through a 90° arc. The TA was then immediately injected with either 0.1 mL of dexamethasone (DM) (0.08mg/kg), saline, nandrolone decanoate (ND) (2mg/kg), or sesame oil. Maximal tetanic tension (Po) was used to measure muscle function on days 1 and 3. To quantify PMN accumulation, a muscle extract was obtained and assayed for myeloperoxidase (MPO) activity and results were calculated as U/mg protein. To visualize PMNs, we performed immunohistochemistry (IH) using antibodies against MPO. The data were analyzed by a one-way ANOVA. RESULTS In a preliminary study, our TA injury led to an immediate decrease in Po from 7.88±1.88N to 5.05±0.85N. As expected, Po continued to drop in all four groups one day after injury. By day 3, however, the DM-treated muscles showed a significant increase in Po (5.68±2.37N) compared to the control group (p = 0.02), while the ND-treated muscle continued to deteriorate (1.56±1.08N) compared to controls (p = 0.03). One day after TA injury both our vehicle-control groups had a doubling of MPO specific activity compared to non-injured muscle. Treatment with DM led to a 55\% increase above controls, while ND treatment led to a 35\% decrease of MPO specific activity compared to controls. By Day 3 the MPO activity had returned to the pre-injury level in all groups. IH showed a similar trend. CONCLUSIONS Contrary to our expectations, we observed a relationship between increased amount of intramuscular PMN on day 1 and the recovery of function after muscle injury on day 3. The data suggest that PMNs may prime the muscle to achieve a faster recovery after injury. Funded in part by NIH (K01HD01165) and a grant from NFL Charities to PGDD

Microbial activity, nutrient dynamics and litter decomposition in a Canadian Rocky Mountain pine forest as affected by N and P fertilizers

To study the specific effects of N and P fertilizers on soil microbial processes under field conditions, a pine forest in southern Alberta was fertilized with ammonium nitrate and urea (0 and 188 kg N ha −1, respectively) singly and in combination with triple super phosphate (0 and 94 kg P ha −1, respectively). Microbial respiration (BR), substrate induced respiration (SIR), metabolic quotient (qCO 2) and rates of microbially mediated key processes were monitored in the forest floor FH material during the growing periods of spring 1990 to fall 1992. A transient increase in soil NH 4 + availability was detected following N addition but significant increases in PO 4 3− availability were detected throughout the study as a result of P enhancement. Microbial variables were unaffected by N addition, whereas soil organic matter and SIR was increased with P fertilization. Microbial BR and qCO 2 were reduced with P fertilization suggesting more efficient utilization of C. Increased net mineralization of P in the P-fertilized plots was found during the second and third summers after fertilization, following immobilization of P during the first year. In contrast, NH 4 +-N mineralization in the N-fertilized plots was significantly increased only during the first year. Rates of nitrification were unaffected by either N or P addition. Decomposition of pine litter was enhanced with N addition only during the first year and P had no effect on decomposition. Addition of N and P at these rates does not appear to impose a major ecological stress to the soil ecosystem.

Responses of soil respiration, soil nutrients, and litter decomposition to inputs from canopy herbivores

We tested whether inputs from canopy herbivores would affect soil processes such as respiration, nutrient cycling, and decomposition along an elevation gradient. The five treatments we used were frass additions, throughfall additions, removal of all litter that fell during the study, removal of greenfall that fell during the study, and controls. Soil respiration was significantly reduced on low and mid elevation sites in litter exclusion, greenfall exclusion and throughfall addition treatments (from 0.846 g CO 2/m 2/h for controls to 0.618, 0.667, and 0.708 g CO 2/m 2/h, respectively, for the three treatments). Throughfall additions containing PO 4 and NH 4 contributed to significant increases in PO 4 (as much as 0.737 mg/l in 100 ml KCl extract greater then controls), but decreases in NO 3, (0.306 mg/l in 100 ml KCl extract less than con trols), in soil solution samples compared to controls. We observed no significant treatment effects on litter decomposition. Precipitation and temperature influenced soil respiration, but both factors showed a significant interaction with elevation. Phosphate concentrations in soil solutions differed significantly with elevation (low elevation mean 0.097 mg/l, mid elevation mean 0.192 mg/l). Elevation had no significant effect on decomposition.

Effect of bovine serum albumin on the calcium release channel of sarcoplasmic reticulum from rabbit skeletal muscle.

The effect of bovine serum albumin (BSA) on the activity of the calcium release channel of the sarcoplasmic reticulum from rabbit skeletal muscle was investigated using both tension recording from skinned fibres and electrophysiological recording of unitary channel currents from planar lipid membranes. BSA had no effect on the Ca2+ affinity of the contractile proteins, elicited no tension per se in Ca(2+)-loaded skinned fibres, but potentiated caffeine-induced tension. Maximum potentiation was observed with 0.05-0.5% BSA. BSA (0.1%) had no detectable effect on the basal activity of the Ca(2+)-release channel incorporated in lipid bilayer. However, channel stimulation elicited by either caffeine (2 mM) or ATP (60 microM) was further enhanced by BSA (0.1%), as indicated by significant increases in Po, the open probability of the channel. These results suggest that BSA can modulate the response of the skeletal muscle SR Ca(2+)-release channel to different activators such as caffeine and ATP.

Effect of mechanical stimulation by tooth brushing on oxygen tension in dog gingiva

To determine oxygen tension ( pO 2) in gingival tissue, an oxygen micro-electrode with a membrane-coated Pt needle was inserted into the gingiva of 12 dogs. Teeth were brushed using a modified Bass technique for 10 s under 200 g pressure. pO 2 increased and reached a maximum 15 min after brushing, then gradually returned to the baseline. A significant increase in pO 2 persisted for approx. 1 h. These findings suggest that short-term stimulation by tooth brushing increases oxygen tension in the gingiva.

Reversal of sexual impotence in male patients with chronic obstructive pulmonary disease and hypoxemia with long term oxygen therapy

Erectile impotence is commonly encountered in male patients with respiratory failure and hypoxia. In this study, 42% of the patients experienced reversal of sexual impotence during long term oxygen therapy (LTOT). We examine the association between sexual impotence, gonadal axis hormones, hypoxia, and oxygen therapy. Nineteen sexually impotent male patients eligible for LTOT ( pO 2 < 7.3 kPa during stable disease) and with sexual impotence received oxygen therapy for 1 month ( n = 12) or 24 h ( n = 7). pO 2, LH, FSH, testosterone, and SHBG (sex hormone binding globulin) were monitored. Five of 12 patients receiving oxygen for 1 month regained sexual potency. The responders showed a significant increase in arterial pO 2 and serum testosterone, and a decline in SHBG compared to non-responders. None of the patients receiving oxygen for 24 h experienced reversal of sexual impotence, despite a significant increase in pO 2. In these patients, serum testosterone did not increase significantly. Reversal of sexual impotence may be achieved in some patients with respiratory failure. The oxygen therapy must, however be administered for an adequate length of time.

Increase in tumor pO2 by perfluorochemicals and carbogen.

The effects of perfluorochemicals (Fluosol-DA) in combination with carbogen (95% O2 and 5% CO2) breathing on the tumor pO2 was investigated. The pO2 in RIF-1 tumors grown in the leg of C3H mice was determined by polarographic method using oxygen microelectrodes with diameters of 50-60 micron. The average and median pO2 in the control RIF-1 tumors was about 13 mm Hg and 6 mm Hg, respectively. Carbogen breathing alone could cause a significant increase in pO2 in some tumors and Fluosol-DA injection (i.v.) alone caused a slight change in tumor pO2. When the animals were treated with both Fluosol-DA injection and carbogen breathing, the pO2 markedly increased in most of the tumors resulting in an average and median tumor pO2 of 80 mm Hg and 60 mm Hg, respectively. Such an increase in tumor pO2 may account for the previous observations by us and others that the response of experimental tumors to radiation could be markedly increased by treating the tumor-bearing animals with carbogen and Fluosol-DA.

Perfluorochemicals and hyperbaric oxygen in radiation therapy

It has been postulated that tumors contain hypoxic cells of decreased radiation sensitivity, which limit curability with radiation therapy. Hyperbaric oxygen has been used in an attempt to improve tumor oxygenation. The nature of the oxygen concentration-radiation sensitivity relationship (oxygen increases the slope of the radiation cell survival curve) suggests that a small number of hypoxic cells, as few as one in one million, would limit tumor curability. Oxygen moves by diffusion from the capillary into the tumor. An increase in partial pressure in the capillary will increase the effective diffusion distance. To improve tissue oxygenation effectively the partial pressure of oxygen in blood must be significantly increased throughout the length of the capillary, in particular at the venous end. Theoretical considerations indicate that hyperbaric oxygen as presently used in radiation therapy, 3 ATA, would lead to only marginal improvement. P arO 2 may be as much as 0.8 atm below that of the inspired gas; this plus the consumption of oxygen along the length of the capillary lead to predictions of values for P End CaO 2 of less than twice normal. Such considerations explain the rather limited success of hyperbaric oxygen with radiation therapy. Thus it is unnecessary to postulate an absence of hypoxic cells to explain this clinical observation. In the presence of perfluorocarbon micelles the non-hemoglobin-bound oxygen carrying capacity of blood is significantly increased. Theoretical considerations predict that the difference between P artO 2 and PO 2 of the inspired gas should be decreased. Furthermore, the nonhemoglobin-bound oxygen carrying capacity should be adequate to satisfy tissue consumption requirements without unloading hemoglobin, thereby avoiding the “PO 2 buffering effect of hemoglobin” and permitting a significant increase in PO 2 throughout the capillary length. This effect has been demonstrated using a rodent tumor model.