Characterisation of steady-state and transient responses of transcutaneouspO2 andpCO2 by mass spectrometry

The paper describes steady-state and transient responses of transcutaneous pO₂ and pCO₂ by mass spectrometry. The effects of factors affecting the responses were estimated from a mathematical model comprising the skin, a membrane of a sampler and a tube connected to a mass spectrometer. The effect of heading temperature, membrane thickness, gas permeabilities of the membrane and the dimensions of the connecting tube were verified experimentally. Transcutaneous tension (tcp) in O₂ in a steady state was much more dependent on temperature and membrane thickness than in CO₂. The transient response of the tcp was found to be improved, without decreasing the magnitude of the steady-state tcp, by a thinner membrane with a lower permeability and by a shorter connecting tube of a larger inner diameter. The method of calibration was also examined because the magnitude of tcp by mass spectrometry was influenced in particular by water vapour.     

Online pH,pCO2 andpO2 monitoring system using two-way cyclic pumping of lactate Ringer as a baseline solution

A system for the online, simultaneous and intermittent analysis of blood pH, pCO₂ pO₂ using two-way cyclic pumping of lactate Ringer as a baseline solution is described. The multi-gas sensor is composed of a pH-ISFET with a temperature sensor, a pH-ISFET-based pCO₂ sensor and a Clark-type pO₂ sensor, bundled in an epoxy rod, and held in place in a flow cell which is directly connectable to an indwelling cannula. The system is controlled by a microcomputer, which supervises pump operation, temperature-compensation of sensor outputs, one-point calibration using sampled blood analysis, and provides graphical display of monitored results. Results of arterial blood-gas monitoring for mongrel dogs are presented which demonstrate the reliability of the present system over extended periods.     

Cell adhesion on phase-separated surface of block copolymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) and poly(dimethylsiloxane)

We investigated the morphological effect of phase-separated block copolymer surfaces composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) and poly(dimethylsiloxane) (PDMS) on protein adsorption and cell adhesion behavior. We observed three different types of phase-separated surface morphologies by TEM and AFM. The elemental composition of phosphorus on the surface increases with the PMPC composition. Furthermore, the polymer surface formed by a block copolymer-containing a higher MPC unit composition shows a slightly lower static water contact angle. This result indicates that the elemental surface ratio of the surface depends on the MPC composition in the block copolymer. Protein adsorption tests revealed that only hydrophobic PDMS domains showed selective protein adsorption. Cell adhesion tests revealed that the number of adhered cells increased with increasing hydrophobic PDMS domain size of block copolymers in serum-containing media. In contrast, no cells adhered onto block copolymer surfaces in serum-free media, whereas a large amount of adhered cells were observed on the hydrophobic PDMS surface. This result indicates that segregated hydrophobic domains on a biocompatible PMPC surface strongly affect serum protein adsorption, thereby promoting considerable cell adhesion, although the surface is hydrophilic. Thus, both the composition of MPC units and the segregated hydrophobic surface morphology are important considerations in biomaterial surface design.     

Choice of polarisation voltage in polarographic oxygen tension measurement: influence ofpCO2 and pH of the medium

The influence of variations in the pH and the pCO₂ of the medium surrounding an oxygen electrode on the polarograms of two types of electrodes was investigated. Most measurements were made with polystyrene-covered monopolar needle electrodes with a 50 μm Pt cathode as used for subcutaneous pO₂ measurements in the foetal scalp during labour. Some of the measurements were made with conventional Clark electrodes with 3 or 25 μm PTFE membranes. It appeared that in an oxygen-free solution the polarisation voltage at the beginning of the hydrogen wave shifted to a less negative value when the pH of the solution was decreased by adding HCl or by increasing the pCO₂. Lowering the pH diminished the flat part of the polarogram. In a fluid with an appreciably high pO₂ the influence of the pH of the solution when changed by adding HCl was negligible, whereas there was a considerable influence of the pC₂ on the polarisation voltage at which the hydrogen wave starts. This may be explained by OH⁻ production through reduction of O₂, which increases pH at the electrode surface. This effect may be undone by CO₂ diffusing rapidly through the membrane covering the Pt surface, but not by the slow influx of H⁺, for which the membrane is much less permeable. Knowledge of these phenomena may be important in choosing the right level of polarisation voltage so that the oxygen electrodes are independent of changes in pCO₂.     

Barley plasma membrane intrinsic proteins (PIP Aquaporins) as water and CO2 transporters

We identified barley aquaporins and demonstrated that one, HvPIP2;1, transports water and CO2. Regarding water homeostasis in plants, regulations of aquaporin expression were observed in many plants under several environmental stresses. Under salt stress, a number of plasma membrane-type aquaporins were down-regulated, which can prevent continuous dehydration resulting in cell death. The leaves of transgenic rice plants that expressed the largest amount of HvPIP2;1 showed a 40% increase in internal CO2 conductance compared with leaves of wild-type rice plants. The rate of CO2 assimilation also increased in the transgenic plants. The goal of our plant aquaporin research is to determine the key aquaporin species responsible for water and CO2 transport, and to improve plant water relations, stress tolerance, CO2 uptake or assimilation, and plant productivity via molecular breeding of aquaporins.     

No effect of trans sodium crocetinate on maximal O(2) conductance or V(O(2),max) in moderate hypoxia

The lumped parameter describing skeletal muscle diffusional conductance for O(2), DM(O(2)), reflects all of the resistances for O(2) in moving from red cell to muscle fiber mitochondria. The purpose of our study was to determine if the carotenoid compound, trans sodium crocetinate (TSC), which has been reported to increase the diffusivity of O(2) in plasma, improves DM(O(2)) and thus, V(O(2),max) in maximally contracting in situ skeletal muscle. V(O(2),max) was measured in the isolated perfused canine gastrocnemius (n=5) during 3 min of isometric tetanic contractions at 1 Hz, while the animal was breathing 12% O(2) (PA(O(2))=32+/-2 Torr, mean+/-S.E.) under two experimental conditions. The first was a control contraction period and the second (following 60 min recovery) was performed within 5 min after infusion of a 0.1 mg x ml(-1) solution of TSC (total dose 100 microg kg(-1)). There were no significant differences in convective O(2) delivery (11.9+/-2.3 vs. 12.1+/-2.2 ml x min(-1) x 100 g(-1)), V(O(2),max) (9.5+/-1.5 vs. 9.6+/-1.5 ml x min(-1) x 100 g(-1)) or calculated DM(O(2)) (0.37+/-0.03 vs. 0.37+/-0.04 ml x min(-1) x 100 g(-1) x Torr(-1)) between contraction periods. As such, our results show that TSC does not improve performance in maximally contracting canine gastrocnemius muscle in situ under moderately hypoxic conditions, suggesting either that TSC in this situation does not increase plasma O(2) diffusivity or that this step in O(2) diffusion from red cell to myocyte does not constrain DM(O(2)).     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

Oxygen tension in rat cerebral cortex microvessels in acute anemia

Polarigraphic microelectrodes were used to study the distribution of oxygen tension (pO(2)) in arterioles (lumen diameters 8-80 microm) and venules (lumen diameters 8-120 microm) in the rat cerebral cortex during acute reductions in blood hemoglobin ([Hb]). Isovolumic hemodilution with 5% albumin solution was performed in steps from an initial [Hb] of 14.1 +/- 0.3 g/dl (control) to 9.8 +/- 0.3 g/dl (step 1), 6.6 +/- 0.4 g/dl (step 2), and 4.6 +/- 0.3 g/dl (step 3). Mild anemia (step 1, hematocrit 30%) led to an increase in pO(2) in the arterial side of the microcirculatory bed, with virtually no change in pO(2) in the venous side. Step 2 (hematocrit 20%) was accompanied by a further insignificant increase in pO(2) in arterioles, while there was a significant reduction (on average to 32 mmHg) in venules. Step 3 (hematocrit 13-14%) led to a (statistically insignificant) increase in pO(2) in arterioles. pO(2) in venules decreased, on average, to 27 mmHg; the proportion of smallest venules with low pO(2) values (less than 20 mmHg) increased to 31% (from 3% in controls). In some capillaries, pO(2) was 5-10 mmHg, which was an indicator of the presence of hypoxic zones in brain tissues. These zones primarily arose close to the smallest capillary and venous microvessels, with slowed or impaired blood flow.     

Direct measurements of oxygen tension at the surface of arterioles,capillaries and venules of the cerebral cortex

Direct pO₂ measurements were made at the surface of arterioles, capillaries, and venules of rabbit brain cortex. The results indicate that a considerable amount of oxygen diffuses through the arteriolar and precapillary wall. Therefore, the arterial blood that reaches the capillaries already has low pO₂ (43±3 mm Hg and about 75%–70% of the blood oxygen capacity).     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.     

HAT2CH2 score performance predicting neurologic events after cardiac implantable electronic device

Objectives: The HAT₂CH₂ score has been evaluated for predicting new-onset atrial fibrillation in several clinical conditions, but never for adverse neurologic events. We aimed to evaluate the effectiveness of HAT₂CH₂ score in predicting neurologic events in patients with cardiac implantable electronic device (CIED), comparing with atrial high-rate episodes (AHRE).Methods: This case-control study enrolled 314 consecutive patients aged 18 years or older with CIED implantation between January 2015 and April 2021. Patient data were analyzed retrospectively. The primary endpoint was subsequent neurologic events (NE) after implantation. AHRE was defined as > 175 bpm (Medtronic®) lasting ≥ 30 seconds. Variables associated with independent risk of NE were identified using multivariate Cox regression analysis with time-dependent covariates.Results: Patients'' median age was 73 years and 61.8% of them were male. During follow-up (median 32 months), 18 NE occurred (incidence rate 2.15/100 patient-years, 95% CI 1.32-4.30). Multiple Cox regression analysis showed that the HAT₂CH₂ score (HR 2.424, 95% CI 1.683 - 3.492, p < 0.001) was an independent predictor for NE. Optimal HAT₂CH₂ score cutoff value was 3 with highest Youden index (AUC, 0.923; 95% CI, 0.881-0.966; p < 0.001). Both AHRE ≥ 1 minute and HAT₂CH₂ score ≥ 3 had the highest AUC of the receiver-operating characteristic (0.898, 95% CI, 0.831-0.965, p < 0.001). Significant increase was observed in NE occurrence rates using the HAT₂CH₂ score (p < 0.001).Conclusion: The HAT₂CH₂ score and episodes of AHRE lasting ≥ 1 minute are independent risk factors for NE in patients with CIED.     

Contribution of P2Y(1) receptors to ADP signalling in mouse spinal cord cultures

Mixed neuronal and glial cell spinal cord cultures from neonates express ADP sensitive P2Y(1,12&13) receptors. ADP (10microM) evoked increases in intracellular calcium that were essentially abolished by the P2Y(1) receptor antagonist MRS2179 (10microM), responses were also absent in preparations from P2Y(1) receptor deficient mice however UTP (100microM) evoked calcium rises were unaffected. ADP also evoked a robust increase in extracellular signal-regulated protein kinase (ERK) phosphorylation that was of similar magnitude in the cultures from wild type and P2Y(1) receptor deficient mice. These results suggest that ADP acts through P2Y(1) receptors to mediate an increase in intracellular calcium but not to stimulate ERK phosphorylation in the spinal cord.     

Attenuation of ethanol-induced ataxia by alpha(4)beta(2) nicotinic acetylcholine receptor subtype in mouse cerebellum: a functional interaction

Many epidemiological studies support the notion that people who drink alcohol also smoke cigarettes and vice versa thereby suggesting a possible functional interaction between these two most widely used psychoactive substances. We have earlier demonstrated that direct intracerebellar (ICB) microinfusion of nicotine dose-dependently antagonizes ethanol-induced ataxia and further that this antagonism occurs in a glutamate-nitric oxide-cyclic guanylyl monophosphate (cGMP) sensitive manner. The present study was designed to determine the possible involvement of specific nicotinic acetylcholine receptor (nAChR) subtype alpha(4)beta(2) in nicotine-induced attenuation of ethanol ataxia. Using the Rotorod test and direct ICB microinfusion technique in stereotaxically cannulated CD-1 male mice, we performed the Rotorod test following ICB administration of the alpha(4)beta(2)-selective agonist, (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403; 31.25, 62.5, 125 ng) on ethanol (2 g/kg; i.p.) ataxia at 15, 30, 45, 60 min post-ethanol injection. RJR-2403 dose-dependently attenuated ethanol ataxia suggesting a role of alpha(4)beta(2) subtype in ameliorating ethanol-induced ataxia. Pretreatment with ICB dihydro-beta-erythroidine (DHbetaE: 125, 250, 500, 750 ng), a potent alpha(4)beta(2)-selective antagonist, significantly reduced RJR-2403's effect further supporting the alpha(4)beta(2) involvement. DHbetaE (ICB) also antagonized ICB nicotine-induced attenuation of ethanol ataxia again reinforcing the role of alpha(4)beta(2) subtype. Additional evidence for the role of alpha(4)beta(2) subtype was provided when ICB alpha(4)beta(2) antisense oligodeoxynucleotide treatment markedly antagonized RJR 2403-induced attenuation of ethanol ataxia compared with missense-treated animals. This was confirmed with an associated decrease in the expression of alpha(4)beta(2) subtypes indicated by immunoblot experiments. In conclusion, the results of the present investigation support an important role of alpha(4)beta(2) nAChR subtype in the expression of nicotine-induced attenuation of ethanol ataxia.     

Effect of H+ and elevated PCO 2on membrane electrical properties of rat cerebral arteries

Some membrane electrical properties of muscle cells from the middle cerebral artery of the rat were recorded with intracellular microelectrodes. The resting membrane potential (E _m) of this preparation was –63 mV. Reduction of extracellular pH to 7.0 in the face of a constantP _{CO 2}of 40 mm Hg had no significant effect onE _m. Similarly the slope of the steady-state voltage/current curves was not different at pH 7.0 compared to control at pH 7.4. In marked contrast, whenP _{CO 2}was elevated to around 60 to 70 mm Hg there was a rapid hyperpolarization and reduction in the slope of the voltage current curve suggesting an increased conductance for one or more ionic species. In addition elevation ofP _{CO 2}increased the slope of theE _m vs. log[K]₀ curve from 46 mV/decade to 59 m V/decade which is in good agreement with a Nernstian potential for a K⁺ selective membrane. These data suggest that while the smooth muscle cells of rat cerebral arteries are relatively insensitive to a small reduction in extracellular pH; reduction of intracellular pH by elevatingP _{CO 2}induces hyperpolarization by increasing K⁺ conductance (g _k). However, it is not clear from these experiments if theP _{CO 2}effects are mediated entirely by changes in pH or if there is a direct membrane action of CO₂.This work is supported by Grant no. HL27862     

Using CHADS2, R2CHADS2, CHA2DS2-VASc score for mortality prediction in patients with abnormal low and high ankle-brachial index

Abnormal low and high ankle brachial index (ABI) is regarded as peripheral artery disease (PAD) which has extremely high morbidity and mortality. How to identify high-risk PAD patients with increased mortality is very important to improve the outcome. CHADS₂, R₂CHADS₂, and CHA₂DS₂-VASc score are clinically useful scores to evaluate the annual risk of stroke in patients with atrial fibrillation. However, there was no literature discussing the usefulness of these scores for cardiovascular (CV) and all-cause mortality prediction in the patients with abnormal ABI. This longitudinal study enrolled 195 patients with abnormal low (< 0.9) and high ABI (> 1.3). CHADS₂, R₂CHADS₂, and CHA₂DS₂-VASc score were calculated for each patient. CV and all-cause mortality data were collected for outcome prediction. The median follow-up to mortality was 90 months. After multivariate analysis, CHADS₂, R₂CHADS₂, and CHA₂DS₂-VASc score were significant predictors of CV and all-cause mortality (all P < 0.001). CHA₂DS₂-VASc score had a better additive predictive value than CHADS₂ and R₂CHADS₂ score for CV mortality prediction. R₂CHADS₂ and CHA₂DS₂-VASc score had better additive predictive values than CHADS₂ score for all-cause mortality prediction. In conclusion, our study is the first study to investigate the usefulness of CHADS₂, R₂CHADS₂, and CHA₂DS₂-VASc score for mortality prediction in patients with abnormal ABI. Our study showed all three scores are significant predictors for CV and all-cause mortality although there are some differences between the scores. Therefore, using the three scoring systems may help physicians to identify the high-risk PAD patients with increased mortality.