Na,K-ATPase alpha isoforms at the blood-cerebrospinal fluid-trigeminal nerve and blood-retina interfaces in the rat (original) (raw)
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Two different Na,K-ATPases in the optic nerve: cells of origin and axonal transport
Proceedings of the National Academy of Sciences, 1984
Two molecular forms of Na,K-ATPase can be isolated from the central nervous system. The two forms can be distinguished by their sensitivities to cardiac glycosides and by the electrophoretic mobilities of their catalytic subunits, a and a(+). Because Na,K-ATPase is a membranebound enzyme, it would be predicted to move in the rapid phase of axonal transport, and this was used as a means to determine which form(s) is made by a defined neuron of the central nervous system. Retinal ganpglion cells were labeled in vivo by intravitreal injection of [3 Simethionine; the Na,K-ATPase that was axonally transported down the optic nerve was purified, and the a and a(+) forms were separated by electrophoresis and detected by fluorography. The two forms were synthesized in the retina in approximately equal amounts. The a(+) form was the predominant form transported from the retinal ganglion cells to the lateral geniculate nucleus and superior colliculus. The oligodendrocytes and other sheath cells of the excised optic nerve, in contrast, synthesized only the a form when incubated in vitro with [35S]methionine. The labeled Na,K-ATPase found at the nerve endings always included a small amount of the a form in addition to the a(+) form. The proportions of the two forms did not change with time after transport, and the presence of labeled a was not affected by infusion of cycloheximide to inhibit intracranial protein synthesis. Hence, although a(+) is the predominant form, the evidence suggests that small amounts of the a form are also made and transported by retinal ganglion cells. The ouabain-inhibited Na,K-ATPase is the enzyme responsible for active transport of Na' and K+ across the cell membrane (1), and is present in both neurons and glia. When isolated from the kidney, it is comprised of a catalytic subunit of Mr approximately 95,000 (a) and a smaller glycoprotein subunit (13) of unknown function. Preparations of Na,K-ATPase from the brain, however, have two biochemically distinct forms of the catalytic subunit, called a and a(+) (2). These differ in electrophoretic mobility in NaDodSO4-containing gels (2-4) and in sensitivity to cardiac glycosides (2, 5, 6). Purified axolemmal membrane from rat brain white matter contains only a(+) (2, 3), while cultured glia contain only a (2), which suggests that a(+) is characteristic of neurons and a, of nonneuronal cells. Neurons of the sympathetic nervous system contain only the a form, however, and synaptosomes from the cerebral cortex contain both forms (2). Although the presence of the a form in synaptosomes may be due to contamination with membrane of glial origin, the evidence is consistent with the hypothesis that neurons can express either form or both. A selective labeling technique is needed to determine which forms are expressed in different neurons of the central nervous system.
Inhibition of Na+/K+-ATPase by Endothelin-1 in Human Nonpigmented Ciliary Epithelial Cells
Journal of Pharmacology and Experimental Therapeutics, 2001
Endothelin-1 (ET-1), a potent vasoconstrictor, lowers intraocular pressure in mammals, either by enhancing the outflow of aqueous humor (AH) via the trabecular meshwork and Schlemm's canal or by reducing AH formation at the ciliary epithelium. Aqueous humor production occurs by passive diffusion of water coupled with active transport of ions, mainly involving Na ϩ :K ϩ :2Cl Ϫ cotransporter and Na ϩ /K ϩ -ATPase pump from serosal to aqueous side. Presently, we have evaluated the effects of ET-1 on Na ϩ :K ϩ :2Cl Ϫ cotransport and Na ϩ /K ϩ -ATPase activity in HNPE cells using 86 Rb ϩ uptake. ET-1 (100 pM-100 nM) decreased mean 86 Rb ϩ uptake by 15% during a 15-min uptake period. ET-1's effect was not prevented by BQ610, an ET A receptor antagonist, but was blocked by
Headache, 2010
Background.—Cerebrospinal fluid sodium concentration ([Na+]csf) increases during migraine, but the cause of the increase is not known.Objective.—Analyze biochemical pathways that influence [Na+]csf to identify mechanisms that are consistent with migraine.Method.—We reviewed sodium physiology and biochemistry publications for links to migraine and pain.Results.—Increased capillary endothelial cell (CEC) Na+, K+, -ATPase transporter (NKAT) activity is probably the primary cause of increased [Na+]csf. Physiological fluctuations of all NKAT regulators in blood, many known to be involved in migraine, are monitored by receptors on the luminal wall of brain CECs; signals are then transduced to their abluminal NKATs that alter brain extracellular sodium ([Na+]e) and potassium ([K+]e).Conclusions.—We propose a theoretical mechanism for aura and migraine when NKAT activity shifts outside normal limits: (1) CEC NKAT activity below a lower limit increases [K+]e, facilitates cortical spreading depression, and causes aura; (2) CEC NKAT activity above an upper limit elevates [Na+]e, increases neuronal excitability, and causes migraine; (3) migraine-without-aura may arise from CEC NKAT over-activity without requiring a prior decrease in activity and its consequent spreading depression; (4) migraine triggers disturb, and treatments improve, CEC NKAT homeostasis; (5) CEC NKAT-induced regulation of neural and vasomotor excitability coordinates vascular and neuronal activities, and includes occasional pathology from CEC NKAT-induced apoptosis or cerebral infarction.
Frontiers in Physiology, 2016
, or the Na + pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β subunit of Na + + 1 , K-ATPase plays an important role in this mechanism because homotypic β 1-β 1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na + pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β 2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na + , K +-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na + pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β 2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β 2 subunit. qPCR results showed a time-dependent increase in the level of β 2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β 2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α 2 subunit in that domain. Our results demonstrate that the apical polarization of Na + , K +-ATPase in RPE cells depends on the expression of the β 2 subunit.
Alterations in the alpha2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2
Proceedings of the National Academy of Sciences of the United States of America, 2005
A number of missense mutations in the Na,K-ATPase alpha2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,K-ATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al. [Vanmolkot, K. R., et al. (2003) Ann. Neurol. 54, 360-366], which we show here to also be functional and kinetically altered. Both mutants have reduced catalytic turnover and increased apparent affinity for extracellular K(+). For both R689Q and M731T, sensitivity to vanadate inhibition is decreased, suggesting that the steady-state E(1) <==> E(2) poise of the enzyme is shifted toward E(1). Whereas the K'(ATP) is not affected by the R689Q replacement, the M731T mutant has an increase in apparent affinity for ATP. Analysis of the structural changes effected by T345A, R689Q, and M731T mutations, based...
Ouabain (OUA) is a cardiac glycoside that binds to Na + ,K +-ATPase (NKA), a conserved membrane protein that controls cell transmembrane ionic concentrations and requires ATP hydrolysis. At nM concentrations, OUA activates signaling pathways that are not related to its typical inhibitory effect on the NKA pump. Activation of these signaling pathways protects against some types of injury of the kidneys and central nervous system. There are 4 isoforms of the alpha subunit of NKA, which are differentially distributed across tissues and may have different physiological roles. Glial cells are important regulators of injury and inflammation in the brain and express the α1 and α2 NKA isoforms. This study investigated the role of α2 NKA in OUA modulation of the neuroinflammatory response induced by lipopolysaccharide (LPS) in mouse primary glial cell cultures. LPS treatment increased lactate dehydrogenase release, while OUA did not decrease cell viability and blocked LPS-induced NF-κB activation. Silencing α2 NKA prevented ERK and NF-κB activation by LPS. α2 NKA also regulates TNF-α and IL-1β levels. The data reported here indicate a significant role of α2 NKA in regulating central LPS effects, with implications in the associated neuroinflammatory processes. Na + ,K +-ATPase (NKA) is an essential membrane protein due to its maintenance of cellular resting potential and osmotic balance 1. NKA requires an ATP molecule to maintain high intracellular K + concentrations and low Na + concentrations, which are important for cellular function and neuronal transmission 2. Functional NKA has α and β subunits 3. The α subunit is the catalytic subunit and the binding site of cardi-otonic steroids, such as ouabain (OUA). All of the NKA subunits have different isoforms 4. The α subunit has 4 isoforms, among which the α1 isoform is expressed in all cells 5. The various isoforms have differing sensitivities to cardiotonic steroids. In mice, the α4 isoform is more sensitive to OUA than the other isoforms, while the α1 isoform is the least sensitive 6. Mutations in the α subunit have been recently reviewed 7 , indicating a role of such mutations in an array of medical conditions, including primary aldosteronism 8 , familial hemiplegic migraine (FHM) 9 , alternating hemi-plegia of childhood (AHC) 10 , cerebellar ataxia, areflexia, pes cavus, optic atrophy, sensorineural hearing loss (CAPOS syndrome) 11 and rapid-onset dystonia-parkinsonism (RDP) 12. Moreover, NKA has a non-pumping function via its action as a signal transducer 13. Xie and Askari 14 demonstrated that NKA activates the Src-Ras-MAPK pathway, which is involved in many cell processes such as growth, apoptosis and adhesion 15–17. NKA also participates in inositol trisphosphate receptor (IP3R) activation, which evokes calcium oscillations by the release of Ca 2+ from the endoplasmic reticulum 18. OUA is extracted from Strophantus gratus and is a hormone produced endogenously by the adrenal gland and the hypothalamus 19. However, its physiological roles remain unclear. OUA inhibits NKA at high doses, thereby inducing an abnormal increase in intracellular Ca +2 and Na + , which triggers apoptosis. High doses of OUA in the central nervous system (CNS) can be used to develop models of mania 20 , while OUA can afford protection at low doses, as evidenced in kidney studies 21, 22 .
Alterations in the α2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2
Proceedings of the National Academy of Sciences, 2005
A number of missense mutations in the Na,K-ATPase α2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,K-ATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al. [Vanmolkot, K. R., et al. (2003) Ann. Neurol. 54, 360-366], which we show here to also be functional and kinetically altered. Both mutants have reduced catalytic turnover and increased apparent affinity for extracellular K + . For both R689Q and M731T, sensitivity to vanadate inhibition is decreased, suggesting that the steady-state E 1 ↔ E 2 poise of the enzyme is shifted toward E 1 . Whereas the K′ ATP is not affected by the R689Q replacement, the M731T mutant has an increase in apparent affinity for ATP. Analysis of the structural changes effected by T345A, R689Q, and M731T mutations, based on homologous repla...