Circadian expression of H,K-ATPase type 2 contributes to the stability of plasma K⁺ levels.

Maintenance by the kidney of stable plasma K(+) values is crucial, as plasma K(+) controls muscle and nerve activity. Since renal K(+) excretion is regulated by the circadian clock, we aimed to identify the ion transporters involved in this process. In control mice, the renal mRNA expression of H,K-ATPase type 2 (HKA2) is 25% higher during rest compared to the activity period. Conversely, under dietary K(+) restriction, HKA2 expression is ∼40% higher during the activity period. This reversal suggests that HKA2 contributes to the circadian regulation of K(+) homeostasis. Compared to their wild-type (WT) littermates, HKA2-null mice fed a normal diet have 2-fold higher K(+) renal excretion during rest. Under K(+) restriction, their urinary K(+) loss is 40% higher during the activity period. This inability to excrete K(+) "on time" is reflected in plasma K(+) values, which vary by 12% between activity and rest periods in HKA2-null mice but remain stable in WT mice. Analysis of the circadian expression of HKA2 regulators suggests that Nrf2, but not progesterone, contributes to its rhythmicity. Therefore, HKA2 acts to maintain the circadian rhythm of urinary K(+) excretion and preserve stable plasma K(+) values throughout the day.

Free-breathing renal magnetic resonance angiography with steady-state free-precession and slab-selective spin inversion combined with radial k-space sampling and water-selective excitation.

The impact of radial k-space sampling and water-selective excitation on a novel navigator-gated cardiac-triggered slab-selective inversion prepared 3D steady-state free-precession (SSFP) renal MR angiography (MRA) sequence was investigated. Renal MRA was performed on a 1.5-T MR system using three inversion prepared SSFP approaches: Cartesian (TR/TE: 5.7/2.8 ms, FA: 85 degrees), radial (TR/TE: 5.5/2.7 ms, FA: 85 degrees) SSFP, and radial SSFP combined with water-selective excitation (TR/TE: 9.9/4.9 ms, FA: 85 degrees). Radial data acquisition lead to significantly reduced motion artifacts (P < 0.05). SNR and CNR were best using Cartesian SSFP (P < 0.05). Vessel sharpness and vessel length were comparable in all sequences. The addition of a water-selective excitation could not improve image quality. In conclusion, radial k-space sampling reduces motion artifacts significantly in slab-selective inversion prepared renal MRA, while SNR and CNR are decreased. The addition of water-selective excitation could not improve the lower CNR in radial scanning.

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

Coronary MR imaging using free-breathing 3D steady-state free precession with radial k-space sampling.

PURPOSE: To investigate the potential of free-breathing 3D steady-state free precession (SSFP) imaging with radial k-space sampling for coronary MR-angiography (MRA), coronary projection MR-angiography and coronary vessel wall imaging. MATERIALS AND METHODS: A navigator-gated free-breathing T2-prepared 3D SSFP sequence (TR = 6.1 ms, TE = 3.0 ms, flip angle = 120 degrees, field-of-view = 360 mm(2)) with radial k-space sampling (384 radials) was implemented for coronary MRA. For projection coronary MRA, this sequence was combined with a 2D selective aortic spin tagging pulse. Coronary vessel wall imaging was performed using a high-resolution inversion-recovery black-blood 3D radial SSFP sequence (384 radials, TR = 5.3 ms, TE = 2.7 ms, flip angle = 55 degrees, reconstructed resolution 0.35 x 0.35 x 1.2 mm(3)) and a local re-inversion pulse. Six healthy volunteers (two for each sequence) were investigated. Motion artifact level was assessed by two radiologists. Results: In coronary MRA, the coronary lumen was displayed with a high signal and high contrast to the surrounding lumen. Projection coronary MRA demonstrated selective visualization of the coronary lumen while surrounding tissue was almost completely suppressed. In coronary vessel wall imaging, the vessel wall was displayed with a high signal when compared to the blood pool and the surrounding tissue. No visible motion artifacts were seen. Conclusion: 3D radial SSFP imaging enables coronary MRA, coronary projection MRA and coronary vessel wall imaging with a low motion artifact level.

Development and maintenance of the neuronal cytoskeleton in aggregated cell cultures of fetal rat telencephalon and influence of elevated K+ concentrations.

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development-dependent expression of several cytoskeletal proteins, including the heavy- and medium-sized neurofilament subunits (H-NF and M-NF, respectively); brain spectrin; synapsin I; beta-tubulin; and the microtubule-associated proteins (MAPs) 1, 2, and 5 and tau protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular beta-tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M-NF preceded that of H-NF, with the latter being detectable only after approximately 3 weeks in culture. Furthermore, MAP 2 and tau protein showed a development-dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron-enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M-NF, H-NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145-kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron-enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton.

Functional roles of Na,K-ATPase subunits

PURPOSE OF REVIEW: Na,K-ATPase is an oligomeric protein composed of alpha subunits, beta subunits and FXYD proteins. The catalytic alpha subunit hydrolyzes ATP and transports the cations. Increasing experimental evidence suggest that beta subunits and FXYD proteins essentially contribute to the variable physiological needs of Na,K-ATPase function in different tissues. RECENT FINDINGS: Beta subunits have a crucial role in the structural and functional maturation of Na,K-ATPase and modulate its transport properties. The chaperone function of the beta subunit is essential, for example, in the formation of tight junctions and cell polarity. Recent studies suggest that beta subunits also have inherent functions, which are independent of Na,K-ATPase activity and which may be involved in cell-cell adhesiveness and in suppression of cell motility. As for FXYD proteins, they modulate Na,K-ATPase activity in a tissue-specific way, in some cases in close cooperation with posttranslational modifications such as phosphorylation. SUMMARY: A better understanding of the multiple functional roles of the accessory subunits of Na,K-ATPase is crucial to appraise their influence on physiological processes and their implication in pathophysiological states

Mejoras de funcionalidades en la gestión de usuariosde la plataforma K-PAX

Esta memoria trata de las mejoras propuestas en la plataforma educativa kPax de la UOC referentes a la gestión de usuarios.

Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

K(+) channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons

Kv3.1 and Kv3.2 K+ channel proteins form similar voltage-gated K+ channels with unusual properties, including fast activation at voltages positive to −10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3.2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1–Kv3.2 current type can have functions other than facilitating high-frequency firing.

Motivational effects of cannabinoids are mediated by μ-opioid and k-opioid receptor

Repeated THC administration produces motivational and somaticadaptive changes leading to dependence in rodents. Toinvestigate the molecular basis for cannabinoid dependenceand its possible relationship with the endogenous opioid system,we explored Δ9-tetrahydrocannabinol (THC) activity in mice lacking μ-, δ- or κ-opioid receptor genes. Acute THCinduced hypothermia, antinociception, and ypolocomotion remained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In contrast, profound phenotypic changes are observed in several place conditioning protocols that reveal both THC rewarding and aversive properties. Absence of μ receptors abolishes THC place preference. Deletion of κ receptors ablates THC place aversion and furthermore unmasks THC place preference. Thus, an opposing activity of μ- and κ-opioid receptors in modulating reward pathways forms the basis for the dual euphoric–dysphoric activity of THC.

Right coronary artery flow velocity and volume assessment with spiral K-space sampled breathhold velocity-encoded MRI at 3 tesla: accuracy and reproducibility.

PURPOSE: To evaluate accuracy and reproducibility of flow velocity and volume measurements in a phantom and in human coronary arteries using breathhold velocity-encoded (VE) MRI with spiral k-space sampling at 3 Tesla. MATERIALS AND METHODS: Flow velocity assessment was performed using VE MRI with spiral k-space sampling. Accuracy of VE MRI was tested in vitro at five constant flow rates. Reproducibility was investigated in 19 healthy subjects (mean age 25.4 +/- 1.2 years, 11 men) by repeated acquisition in the right coronary artery (RCA). RESULTS: MRI-measured flow rates correlated strongly with volumetric collection (Pearson correlation r = 0.99; P < 0.01). Due to limited sample resolution, VE MRI overestimated the flow rate by 47% on average when nonconstricted region-of-interest segmentation was used. Using constricted region-of-interest segmentation with lumen size equal to ground-truth luminal size, less than 13% error in flow rate was found. In vivo RCA flow velocity assessment was successful in 82% of the applied studies. High interscan, intra- and inter-observer agreement was found for almost all indices describing coronary flow velocity. Reproducibility for repeated acquisitions varied by less than 16% for peak velocity values and by less than 24% for flow volumes. CONCLUSION: 3T breathhold VE MRI with spiral k-space sampling enables accurate and reproducible assessment of RCA flow velocity.

[Archives de la Parole]. , La parabole de l'enfant prodigue : récit (dravidien, malayālam, province de Madras) / [Sir George Grierson], éd. ; Linguistic survey of India ; [C.K. Gopalamenon], voix

[Traditions. Asie. Inde. Province de Madras [i.e. Chennai]. État du Kérala. Région du Malabar]