CO2 fixation in the nervous tissue IV. CO2 fixation and citrate metabolism in lobster nerve

Summary The specific activity of citric acid of the lobster nerve which was incubated in Ringer-bicarbonate solution containing ¹⁴C-bicarbonate was determined. The specific activity of citric acid was higher than that of malic acid by a factor of 2.5. The citric acid obtained from the lobster nerve was degraded with an improved method which is described in this paper. The ratio of the radio-activity of C-6 to C-1 of citric acid was 11. Aspartic acid obtained from the lobster nerve was also degraded, and the ratio of the radioactivity of C₄ to C₁ was almost 101.From these results, it is assumed that CO₂ fixation in the lobster nerves occurs at the oxalosuccinate level and at the oxaloacetate level and that the rates of these fixations were almost the same. Thus the active backwards reaction from -ketoglutaric acid to citric acid in lobster nerve was confirmed. It was also possible that both the activity of the citrate cleavage enxyme and the mixing of the dicarboxylic acid carboxyl groups were minimal.The concentration of oxaloacetic acid was estimated to be 2 mmole/mg protein, or 4 mmoles for a 50 mg nerve.Fellow of the Rockefeller Foundation     

Fluoxetine augments ventilatory CO2 sensitivity in Brown Norway but not Sprague Dawley rats

The Brown Norway (BN; BN/NHsdMcwi) rat exhibits a deficit in ventilatory CO₂ sensitivity and a modest serotonin (5-HT) deficiency. Here, we tested the hypothesis that the selective serotonin reuptake inhibitor fluoxetine would augment CO₂ sensitivity in BN but not Sprague Dawley (SD) rats. Ventilation during room air or 7% CO₂ exposure was measured before, during and after 3 weeks of daily injections of saline or fluoxetine (10 mg/(kg day)) in adult male BN and SD rats. Fluoxetine had minimal effects on room air breathing in BN and SD rats (p > 0.05), although tidal volume (V_T) was reduced in BN rats (p < 0.05). There were also minimal effects of fluoxetine on CO₂ sensitivity in SD rats, but fluoxetine increased minute ventilation, breathing frequency and V_T during hypercapnia in BN rats (p < 0.05). The augmented CO₂ response was reversible upon withdrawal of fluoxetine. Brain levels of biogenic amines were largely unaffected, but 5-HIAA and the ratio of 5-HIAA/5-HT were reduced (p < 0.05) consistent with selective and effective 5-HT reuptake inhibition. Thus, fluoxetine increases ventilatory CO₂ sensitivity in BN but not SD rats, further suggesting altered 5-HT system function may contribute to the inherently low CO₂ sensitivity in the BN rat.     

Water and total CO2 reabsorption along the rat proximal convoluted tubule

Micropuncture experiments were performed on rat kidney to evaluate the profile of water and total CO₂ reabsorption along the proximal tubule. Three to eight samples were collected along the same nephron and the puncture-to-glomerulus distances were measured for each site. In Munich rats with accessible glomeruli, the water reabsorption rate was found to be constant all along the first five millimeters of proximal tubule. In Sprague Dawley rats with no accessible glomerulus, the same observation was made for these five millimeters, and the water reabsorption rate per mm along this segment was found to be a function of the glomerular filtration rate. For the two last millimeters of tubule accessible at the kidney surface, the water reabsorption rate was found to decrease in 5 out of the 21 tubules studied and ranged from 0.15–3.5 nl·min^–1·mm^–1.In Sprague Dawley rats the fall in the luminal total CO₂ concentration (CO₂)_t along the tubular length was nearly constant (21 mmole·l^–1) between Bowman's capsule and the end proximal tubule, irrespective of the plasma (CO₂)_t value. The distance needed to reach half-maximum total CO₂ reabsorption varied from 1.1–1.9 mm from one tubule to another, as a function of the total CO₂ filtered load. These data suggest that the tubular length involved in avid bicarbonate reabsorption increases as a function of the filtered load and that in the first millimeters of tubule, bicarbonate reabsorption depends on a rapidly saturable mechanism. However, no close relationship was found between total CO₂ movement or the calculated transepithelial chloride gradient on the one hand and water reabsorption along the convoluted proximal tubule on the other.     

Ultrastructural analysis of TiO2 nanotubes with photodecomposition of water into O2 and H2 implanted in the nude mouse

To analyze the biocompatibility and O2 generation of TiO2 nanotubes via photodecomposition of water into O2 and H2 in vivo, samples were implanted under the inguinal skin of the nude mouse. Venous oxygen saturation (SvO2) of the inguinal skin over the implanted region was measured with a tissue oximeter and the ultrastructures were examined with an electron microscope. Four weeks after the implantation, SvO2 of the inguinal skin of the groups with TiO2 nanotubes was 30-40% higher than that of the opposite control region (54%). SvO2 of the other groups, comprising splenic autografts, fetal cardiac tissue transplantation and surgical procedure without TiO2 nanotubes, was roughly the same as that of controls. Ultrastructurally, TiO2 nanotubes were phagocytized by the macrophage and promoted filament formation in its cytoplasm. Neither death of the cell nor destruction of the tissue was recognized. These findings indicate excellent biocompatibility and O2 generation of TiO2 nanotubes in vivo.     

Activation of the CB2 receptor system reverses amyloid-induced memory deficiency

Cannabinoid type 2 (CB₂) agonists are neuroprotective and appear to play modulatory roles in neurodegenerative processes in Alzheimer's disease. We have studied the effect of 1-((3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl) carbonyl) piperidine (MDA7)—a novel selective CB₂ agonist that lacks psychoactivity—on ameliorating the neuroinflammatory process, synaptic dysfunction, and cognitive impairment induced by bilateral microinjection of amyloid-β (Aβ)_1–40 fibrils into the hippocampal CA1 area of rats. In rats injected with Aβ_1–40 fibrils, compared with the administration of intraperitoneal saline for 14 days, treatment with 15 mg/kg of intraperitoneal MDA7 daily for 14 days (1) ameliorated the expression of CD11b (microglia marker) and glial fibrillary acidic protein (astrocyte marker), (2) decreased the secretion of interleukin-1β, (3) decreased the upsurge of CB₂ receptors, (4) promoted Aβ clearance, and (5) restored synaptic plasticity, cognition, and memory. Our findings suggest that MDA7 is an innovative therapeutic approach for the treatment of Alzheimer's disease.     

Differential distribution of γ-aminobutyric acidA, receptor subunit (α1, α2, α3, α5 and β2+3) immunoreactivity in the medial prefrontal cortex of the rat

A detailed mapping of the γ-aminobutyric acid (GABA)_A receptor subunits (α₁, α₂, α₃ and β₂₊₃) in the infralimbic/ventral prelimbic region (IL/vPL) of the rat frontal cortex was carried out using subunit-specific antibodies. The α₁ and β₂₊₃ subunit antibodies immunostained all layers of the IL/vPL region. Layers II and III displayed immunostaining of cell bodies whereas I, V and VI showed predominantly neuropil staining. The size of the α₁-positive cell bodies corresponded to that of small interneurons (range, 20–55 μm²; mean ± SEM, 37 ± 5.5 μm²) as well as pyramidal cells or large interneurons (range, 87–135 μm²; mean ± SEM, 103.4 ± 9.7 μm²). However, β₂₊₃ antibody immunostained only small cell bodies. Immunoreactivity for α₂ was restricted to layers I and II, whereas α₃ and α₅ subunit expression was seen only in layer VI. The antibody to the α₂ subunit immunostained small cell bodies (range, 29–63 μm²; mean ± SEM, 32 ± 4.5 μm²) in layer II, resembling interneurons. Conversely, both α₃ and α₅ antibodies immunostained large cell bodies (range, 94–151 gmm²; mean ± SEM, 115.7 ± 13.4 μm²), consistent with pyramidal cell labelling in layer VI.     

Development of ZrO2/SiO2 bioinert ceramic coatings for biomedical application

Gas tunnel-type plasma spraying (GTPS) was employed to deposit ZrO₂/SiO₂ bioinert ceramic composite coatings with an appropriate thickness on SUS 304 substrate. Zirconia and fused silica powders, with equal wt%, have been mixed together in ceramic pot for 30 min and internally fed in the plasma jet. The composite coatings were sprayed at two different gas flow rates (120 and 150 l/min) and constant vortex arc current of 450 A and gun current of 50 A. The microstructure of as-sprayed coatings was examined by scanning electron microscope. Elemental analysis was achieved for the composite coatings using EDS analysis unit which is attached to SEM. Phase structure was investigated by X-ray diffraction. The hardness and abrasive wear test of the coatings were investigated. The biological property of the coatings was examined by immersing the as-sprayed coatings in simulated body fluid (SBF) solution for 20 days at 36.5 C. The growth of apatite (HA) on the coatings surfaces was observed by SEM and EDX analysis.     

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe₃O₄ and Fe₃O₄@SiO₂ NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe₃O₄ NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe₃O₄ NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO₂ shell, Fe₃O₄@SiO₂ NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe₃O₄@SiO₂ NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.     

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M₁ receptors negatively modulates the activity of inhibitory muscarinic M₂ receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involved endogenous adenosine tonically activating A₁ receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [³H]-choline (2.5 μCi/ml). Selective activation of muscarinic M₁ and adenosine A₁ receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R-N⁶-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [³H]-ACh release induced by muscarinic M₂ receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M₁ autoreceptors, suggesting that suppression of M₂-inhibiton by A₁ receptor activation is independent on muscarinic M₁ receptor activity. Conversely, the negative crosstalk between M₁ and M₂ autoreceptors seems to involve endogenous adenosine tonically activating A₁ receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A₁ receptors at the rat motor endplate.     

CO2 retention in lung disease; could there be a pre-existing difference in respiratory physiology

Some patients with lung disease retain CO₂, while others with similar lung function do not. This could be explained if CO₂ retainers had a pre-existing low hypercapnic ventilatory response (HCVR) and, from this, a tendency to retain CO₂. To test if such a phenomenon exists in healthy people, we determined the change in end-tidal P_CO2 (ΔPET_CO2) produced by the addition of a dead-space (DS), during wakefulness and sleep, and related this to the HCVR measured awake. The group mean (n=14) HCVR slope was 2.2±1.1 (S.D.) L min⁻¹ mmHg⁻¹. The ΔPET_CO2 with the application of DS was 1.6±1.6 mmHg awake and 2.6±2.2 mmHg asleep. During wakefulness the ?PET_CO2 with DS did not correlate with the HCVR slope. However, during sleep, four subjects had a marked increase in the ΔPET_CO2 (3.7, 4.3, 6.2, 8.0 mmHg) and a relatively low HCVR (slope 1.5, 1.7, 1.5, 1.7 L min⁻¹ mmHg⁻¹, respectively). We speculate that such individuals, should they develop lung disease, may be predisposed to retain CO₂.     

Chronic hypoxia suppresses the CO2 response of solitary complex (SC) neurons from rats

We studied the effect of chronic hypobaric hypoxia (CHx; 10–11% O₂) on the response to hypercapnia (15% CO₂) of individual solitary complex (SC) neurons from adult rats. We simultaneously measured the intracellular pH and firing rate responses to hypercapnia of SC neurons in superfused medullary slices from control and CHx-adapted adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. We found that CHx caused the percentage of SC neurons inhibited by hypercapnia to significantly increase from about 10% up to about 30%, but did not significantly alter the percentage of SC neurons activated by hypercapnia (50% in control vs. 35% in CHx). Further, the magnitudes of the responses of SC neurons from control rats (chemosensitivity index for activated neurons of 166 ± 11% and for inhibited neurons of 45 ± 15%) were the same in SC neurons from CHx-adapted rats. This plasticity induced in chemosensitive SC neurons by CHx appears to involve intrinsic changes in neuronal properties since they were the same in synaptic blockade medium.     

Effect of chronic activation of 5-HT3 receptors on 5-HT3, 5-HT1A and 5-HT2A receptors functional activity and expression of key genes of the brain serotonin system

Among serotonin (5-HT) receptors, the 5-HT₃ receptor is the only ligand-gated ion-channel. Little is known about the interaction between the 5-HT₃ receptor and other 5-HT receptors and influence of 5-HT₃ chronic activation on other 5-HT receptors and the expression of key genes of 5-HT system. Chronic activation of 5-HT₃ receptor with intracerebroventricularly administrated selective agonist 1-(3-chlorophenyl)biguanide hydrochloride (m-CPBG) (14 days, 40 nmol, i.c.v.) produced significant desensitization of 5-HT₃ and 5-HT_1A receptors. The hypothermic responses produced by acute administration of selective agonist of 5-HT₃ receptor (m-CPBG, 40 nmol, i.c.v.) or selective agonist of 5-HT_1A receptor (8-hydroxy-2-(di-n-propylamino)tetralin) (8-OH-DPAT, 1 mg/kg, i.p.) was significantly lower in m-CPBG treated mice compared with the mice of control groups. Chronic m-CPBG administration failed to induce any significant change in the 5-HT_2A receptor functional activity and in the expression of the gene encoding 5-HT_2A receptor. Chronic activation of 5-HT₃ receptor produced no considerable effect on the expression on 5-HT₃, 5-HT_1A, and 5-HT transporter (5-HTT) and tryptophan hydroxylase-2 (TPH-2) genes – the key genes of brain 5-HT system, in the midbrain, frontal cortex and hippocampus. In conclusion, chronic activation of ionotropic 5-HT₃ receptor produced significant desensitization of 5-HT₃ and postsynaptic 5-HT_1A receptors but caused no considerable changes in the expression of key genes of the brain 5-HT system.     

Gadolinium complex and phosphorescent probe-modified NaDyF4 nanorods for T1- and T2-weighted MRI/CT/phosphorescence multimodality imaging

To compensate for the deficiencies of individual imaging modalities, lanthanide-based nanoparticles are ideal building blocks for multifunctional contrast agents. Herein, oleic acid-coated NaDyF₄ nanorods (DyNPs) were synthesized by the hydrothermal method, and then coated with α-cyclodextrin (α-CD) and modified with gadolinium complex (Gd-DTPA) to obtain hydrophilic and functionalized nanoparticles (DyNPs-Gd). By loading the phosphorescent probe (iridium-complex) within the surface hydrophobic layer, the developed nanophosphors (DyNPs-Gd-Ir) could be further applied in phosphorescent cell labeling. The Dy in the host induces a high X-ray absorption ability for X-ray computed tomography (CT) and negative enhancement for T₂-weighted magnetic resonance imaging (MRI), whereas positive contrast for T₁-weighted MRI results from the Gd-DTPA. DyNPs-Gd-Ir has been successfully applied to T₁- and T₂-weighted MRI/CT in vivo. Toxicity studies demonstrated that DyNPs-Gd-Ir exhibited low toxicity to living systems. Therefore, DyNPs-Gd-Ir could be a platform for next-generation contrast agents for T₁- and T₂-weighted MRI/CT/phosphorescence multimodal imaging.     

C9orf72 G4C2 repeat expansions in Alzheimer's disease and mild cognitive impairment

C9orf72 G₄C₂ repeat expansion is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Its role in Alzheimer's disease (AD) is less clear. We assessed the prevalence of G₄C₂ pathogenic repeat expansions in Flanders-Belgian patients with clinical AD or mild cognitive impairment (MCI). In addition, we studied the effect of non-pathogenic G₄C₂ repeat length variability on susceptibility to AD, and on AD cerebrospinal fluid (CSF) biomarker levels. A pathogenic repeat expansion was identified in 5 of 1217 AD patients (frequency <1%). No pathogenic expansions were observed in patients with MCI (n = 200) or control individuals (n = 1119). Nonpathogenic repeat length variability was not associated with AD, risk of conversion to AD in MCI individuals, or CSF biomarker levels. We conclude that pathogenic C9orf72 G₄C₂ repeat expansions can be detected in clinical AD patients and could act as a contributor to AD pathogenesis. Non-pathogenic repeat length variability did not affect risk of AD or MCI, nor AD biomarker levels in CSF, indicating that C9orf72 is not a direct AD risk factor.     

Bifeprunox and aripiprazole suppress in vivo VTA dopaminergic neuronal activity via D2 and not D3 dopamine autoreceptor activation

Bifeprunox and aripiprazole are two novel antipsychotics presenting partial agonistic activity for the D₂ and D₃ dopamine (DA) receptors. Using in vivo electrophysiological paradigms in anaesthetized rats, we have previously shown that both drugs independently inhibit the spontaneous firing and bursting activity of ventral tegmental area (VTA) dopaminergic neurons and partially reverse the suppressing effect of the full DA receptor agonist apomorphine. Moreover, we have also shown that the D_2/3 receptor antagonist haloperidol prevents the inhibitory effects of these antipsychotics, confirming their partial D₂-like agonistic activities [L. Dahan, H. Husum, O. Mnie-Filali, J. Arnt, P. Hertel, N. Haddjeri, Effects of bifeprunox and aripiprazole on rat serotonin and dopamine neuronal activity and anxiolytic behaviour, J. Psychopharmacol. (2009)]. In the present electrophysiological study, selective antagonists of D₂ and D₃ receptors were used to further characterize the inhibitory role of bifeprunox and aripiprazole on the D₂ and D₃ receptors in vivo. Administration of bifeprunox (250 μg/kg, i.v.) or aripiprazole (300 μg/kg, i.v.) reduced the firing activity of VTA DA neurons by 40–50%. The bursting activity was reduced by 95% and 77% by bifeprunox and aripiprazole, respectively. Systemic administration of the preferential D₃ receptor antagonist GR218,231 (200 μg/kg, i.v.) did not modify the inhibitory effect of bifeprunox or aripiprazole, either on the firing or on the bursting activity. On the other hand, the preferential D₂ receptor antagonist L741,626 (500 μg/kg, i.v.) completely blocked the inhibitory effect of both bifeprunox and aripiprazole on the VTA DA neuronal activity. The present study shows that bifeprunox and aripiprazole behave as partial D₂, but not D₃, receptor agonists in vivo, inhibiting the firing activity (preferentially the phasic activity) of VTA DA cells.     

Activation of imidazoline I2B receptors is linked with AMP kinase pathway to increase glucose uptake in cultured C2C12 cells

Guanidine, the active ingredient extracted from Galega officinalis, is introduced as a ligand for imidazoline I₂ receptor (I₂R) because guanidine decreased plasma glucose via an activation of I_2BR to increase glucose uptake into skeletal muscle isolated from Wistar rats. However, the signals for this action of guanidine remained obscure. In the present study, we used the cultured skeletal muscle fibroblast named C₂C₁₂ cell line to investigate this point. We found that guanidine increased the phosphorylation of AMP-activated protein kinase (AMPK) in addition to the higher of glucose transporter GLUT4 expression and glucose uptake. These effects of guanidine were blocked by the pretreatment with I₂R antagonist BU224 but not by the blockade of I_2AR amiloride. Moreover, compound C at concentrations sufficient to inhibit AMPK blocked the guanidine-induced glucose uptake and GLUT4 protein level. These results suggested that guanidine increases glucose uptake via an activation of I_2BR through AMPK activation in skeletal muscle cell; this view has not been mentioned before.