ATPase Transport System Research Articles

Effects of hypoxia induced by Na 2S 2O 4 on intracellular calcium and resting potential of mouse glomus cells

Isolated and cultured glomus cells, obtained from mouse carotid bodies, were superfused with Ham's F-12 equilibrated with air (mean PO 2, 119 Torr; altitude 1350 m). [Ca 2+] o was 3.0 mM. In one experimental series, dual cell penetrations with microelectrodes measured intracellular calcium ([Ca 2+] i) and the resting potential ( E m). In another series, [Ca 2+] i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean E m of −42.4 mV and [Ca 2+] i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential ( E Ca) was 137±0.74 mV. Hypoxia, induced by Na 2S 2O 4 1 mM, reduced pO 2 to 10–14 Torr. This effect was accompanied by cell depolarization to −19.1 mV. Hypoxia increased [Ca 2+] i to 231 nM when detected with Ca-sensitive microelectrodes, but only to 130.2 nM when measured with Indo-1/AM. Calcium increases were preceded by decreases in [Ca 2+] i, which also were more pronounced with microelectrode measurements. CoCl 2 1 mM blocked the hypoxic [Ca 2+] i increase and exaggerated the decreases in [Ca 2+] i. Correlations between Δ E m and Δ[Ca 2+] i during hypoxia were significant ( p<0.05) in 19% of the cells. But, in 29% of them significance was at the p<0.1 level. In the rest (52%), there was no correlation between these parameters. Thus, voltage-gated calcium channels are rare in mouse glomus cells. Their activation by depolarization cannot explain the two to threefold increase in [Ca 2+] i seen during hypoxia. More likely, [Ca 2+] i increase may be due to hypoxic inactivation of a Ca–Mg ATPase transport system across the cell membrane. The blunting of hypoxic [Ca 2+] i increase, seen in Indo-1/AM experiments, is probably due to its solvent (DMSO), which also depresses hypoxic cell depolarization.

Role of Calcium/Calmodulin-Mediated Processes in Protozoa

Publisher Summary This chapter describes the roles of calcium and calmodulin (CaM) in various protozoan systems and examines the structure of Tetrahymena CaM, its localization and cytology, and the regulation of specific enzyme systems. The presence of CaM in protozoa was first reported in Tetrahymena. While the structure of CaM is conserved in nature, Tetrahymena CaM possesses several unique structural characteristics that account for the biochemical and functional properties peculiar to this ciliate. CaM is a multifunctional protein with a molecular mass of 16,680 containing 148 amino acids, a high proportion of which are acidic residues. Following the stimulation of a cell, there is an increase in the intracellular free Ca2+ concentration due to the movement of Ca2+ through channels in the plasma membrane or due to its release from intracellular organelles. Each CaM molecule can bind as many as four Ca2+. The binding of Ca2+ produces conformational changes to a more helical structure that exposes hydrophobic regions, which, in turn, can bind to and activate CaM-binding proteins. The CaM–Ca2+complex can function directly and rapidly on an effector system, such as the ATPase transport system, or indirectly and slowly on a regulatory system, such as a protein kinase.

EXTRA- AND INTRACELLULAR MG AND ELECTROLYTE CONCENTRATIONS DURING PREGNANCY AND AT BIRTH: FETO-MATERNAL RELATIONSHIP

During pregnancy, Mg deficiency may play a role in pathological states such as uteroplacental insufficiency, gestosis, premature delivery and low birth weight babies. A longitudinal study was undertaken to establish the normal values of the following parameters in maternal and mixed cord blood : plasmatic concentration of Na, K, P, Ca, Mg and protein and intracellular concentration of Na, K, Mg in lymphocytes and erythrocytes. During pregnancy, the previously described fall of total serum Mg concentration was found. The mean intracellular Mg however did not change significantly. However, in 10/35 women, intraerythrocyte and lymphocyte Mg concentration had a tendency to fall. At birth and for every studied extracellular ion but Na, there was a concentration gradient mother-cord indicating the role of the placenta in ionic transfers. In erythrocytes, there was a significant correlation between mother and cord blood for each ion. In lymphocytes, this correlation was only present for Mg. Furthermore, there was a constant correlation between Mg and K concentration in both erythrocytes and lymphocytes. This could be related to effect of Mg ions on cell membranes and/or the Na/K ATPase transport system.

Genetic alteration in the (Na+ + K+) ATPase transport system expressed in human lymphoblasts and their isolated plasma membranes.

A series of ouabain-resistant human lymphoblastoid lines were shown to be genetically altered in the (Na+ + K+) ATPase transport system. The sensitivity to ouabain of Rb+ uptake in intact cells and (Na+ + K+) dependent ATP hydrolysis in purified plasma membrane vesicles was compared with measurement of ouabain binding to intact cells. Preliminary evidence for at least two categories of ouabain resistance was obtained.

Prostaglandin F 2α and insulin stimulate phosphate uptake and (Na +, K +)ATPase activity in resting mouse fibroblast cultures

The (Na +, K +)ATPase transport system in resting 3T3 Swiss mouse fibroblasts is rapidly activated by prostaglandin F 2α and insulin, which initiate DNA synthesis in these cells. Prostaglandin F 2α, but not insulin, promotes a rapid increase in P i uptake which is partially coupled to the Na + pump. This rapid activation of both transport systems occurs by a mechanism which does not require fluctuation of cyclic AMP levels or new protein synthesis. A subsequent protein synthesis-dependent increase in P i uptake is stimulated by insulin and prostaglandin F 2α. These results suggest that different types of control of membrane transport occur during growth stimulation.

Ouabain-resistant human lymphoblastoid lines altered in the (Na+ + K+)-dependent ATPase membrane transport system.

Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)-dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 muM ouabain. Ouabain-resistant (OUA(R)) cells with normal growth at 50 times this dose were recovered at a frequency 1 X 10(-6). This frequency was increased 9-fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR-191, under conditions where both increased the frequency of 8-azaguanine-resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUA(R) clones show showed 30-50% of the wild type amount of 3H-ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 muM at 0.5 mM K+, as observed in wild-type cells. Both the initial rate of uptake of 86Rb+ in OUA(R) cells and the (Na+ + K+)-dependent ATPase activity of OUA(R) plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUA(R) cells in the absence of ouabain were unchanged compared with wild type cells.

The Regulation of Stomatal Aperture in Tobacco Leaf Epidermal Strips II. The Effect of Ouabain

The K+-dependent, light-stimulated opening of stomata on tobacco and V icia faba epidermal strips was found to be rapidly reduced by low concentrations of ouabain. On removing ouabain stomatal aperture rapidly increased. This suggests that the influx of K+ into the guard cells is associated with a membrane�bound transport ATPase. Experiments with 10-5M p.chloromercuribenzoate (PCMB) and ouabain indicate that the considered transport ATPase is not markedly affected by PCMB. The stomatal opening obtained in the presence of Na+ alone is also decreased on the addition of ouabain, though ouabain does not prevent the longer-term stomatal opening which occurs in the dark in the presence of N a + alone. In the light, recovery of stomatal opening on the removal of ouabain from the bathing medium only occurred in the presence of K+. It is considered that an ATPase�linked K+ transport system could give the rapid rate of influx that would be necessary to bring about stomatal opening in the times observed. The presence of an ATPase transport system would give an evolutionary link between the stomatal control mechanism and that associated with the function of other excitable cells such as nerve and muscle.