EFFECT OF THE SERUM CONCENTRATION OF THE GROWTH MEDIUM ON THE SODIUM PUMP SITE DENSITY OF CULTURED HeLa CELLS

Aiton, James F.; Lamb, J. F.

doi:10.1113/expphysiol.1984.sp002799

Cited by 11 publications

(13 citation statements)

References 35 publications

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“…It is therefore not surprising that the activities of both these systems are closely interrelated (16)(17)(18)(19)(20)(21)(22). Besides the concentration of Na' affecting the activity of the Na',K+-ATPase, under certain conditions that lead to increases in Na+ content there is an associated increase in the number of Na+,K+-ATPase molecules on the plasma membrane (16,(23)(24)(25)(26)(27)(28)(29)(30) and an increase in the abundance of their corresponding mRNA (29,30). This has led to the idea that the Na' concentration itself may have a pretranslational effect resulting in these reported increases (27,31).…”

mentioning

confidence: 99%

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Qian

Pastor‐Anglada

Englesberg

1991

Proc. Natl. Acad. Sci. U.S.A.

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Previous work suggested that the structural gene for the A system transporter and the mRNA for the a subunit of the Na+,K+-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance ofthe a, subunit of Na',K'-ATPase mRNA over that found in repressed WT. The level of Na+ per cell in alar4 was not significantly greater than that found in the WT. To further characterize the likely coregulation of both genes, we have studied the A system activity and Na+,K+-ATPase mRNA a,-subunit levels in cells grown under various conditions that result in repression or derepression of the A system in the WT. System A activity increased up to 2-3 times the basal transport rate (repressed state) and Na+,K+-ATPase mRNA a,-subunit levels showed a 3-fold increase after amino acid starvation (derepressed state). These changes occurred along with a decrease in intracellular Na+ levels. N-Methyl-a-aminoisobutyric acid and fi-alanine, previously shown to be corepressors for the A system, prevented to a similar extent A system derepression and Na+,K+-ATPase mRNA a,-subunit accumulation. On the other hand, phenylalanine and lysine, amino acids that are not corepressors of the A system, failed to significantly prevent derepression of both genes. Hybrids between the WT and ala'4 have the phenotype of the WT when grown under repressed conditions. These results give further support to the proposition that both the A system transporter and mRNA for the aI subunit of the Na+,K+-ATPase are coordinately controlled by regulatory gene R1 and elevated Na+ concentrations are not involved. No Na+,K+-ATPase activity was detected in derepressed cells. Activity was restored by the addition of monensin. However, this activity was no greater than that obtained in repressed cells. Indications are that the reduced Na+ content in derepressed cells inhibits Na+,K+-ATPase activity and that conditions that favored derepression do not allow for de novo synthesis of the Na+,K+-ATPase.Evidence has been presented suggesting that the activity of the A system of amino acid transport and the Na+,K+-ATPase is coordinately regulated at the transcriptional level by means of repression and negative control by the A system regulatory gene R1 (1).The A system and the Na+ ,K+-ATPase are ubiquitous integral membrane proteins in mammalian cells that function as secondary and primary active transporters, respectively. The A system accepts as substrates most neutral amino acids but shows a strong preference for proline and small straight-chain amino acids such as alanine (2). It is Na' dependent and transports one Na' into the cells with each amino acid (3). This system is inducible by hormones (4-6), repressible by amino acids, which it generally transports (7-11), and present with increased activ...

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mentioning

confidence: 99%

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Qian

Pastor‐Anglada

Englesberg

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…CHO-K1 (WT), alar4, medium employed in this study, MEMCHO-4, and cultural conditions have been described (1,3,7 [20][21][22][23][24][25][26][27][28][29][30] sec. The dishes were allowed to dry, 0.6 ml of 0.1 M NaOH was added, and after 1 hr a sample of 0.4 ml was removed for the determination of radioactivity.…”

Section: Methodsmentioning

confidence: 99%

“…There is evidence that conditions leading to increases in intracellular Na' are associated with an increase in Na',K+-ATPase activity (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In some cases, these increases in activity have been shown to be the result of increases, probably reversible, in abundance of this enzyme (17,18,21,22,24,(28)(29)(30)(31) and the corresponding mRNA (30,31).…”

mentioning

confidence: 99%

“…In some cases, these increases in activity have been shown to be the result of increases, probably reversible, in abundance of this enzyme (17,18,21,22,24,(28)(29)(30)(31) and the corresponding mRNA (30,31). The increase in just the activity of the Na+,K+-ATPase per cell, without any accompanying increase in abundance, can reasonably be explained on the basis that the intracellular Km of the pump for Na' is about the concentration ofcytosolic Na+ under "normal" conditions (21).…”

mentioning

confidence: 99%

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alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

Qian¹,

Jones²,

McDonough³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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A constitutive mutant, alar4, for the A system of amino acid transport, has increased activity and amount of the A system. This is accompanied by increased sensitivity to ouabain, as measured by efficiency of plating, and increased activity and abundance of the Na+,K+-ATPase that is present in the parental cell line, CHO-K1 (wild type). The latter was shown by increases in (i) ouabain-inhibitable "Rb uptake in intact cells, (ii) ouabain-inhibitable ATPase activity in mixed membrane vesicles, and (iii) number of ouabain-binding sites and by similar Kd values for ouabain binding and Ki12 for ouabain inhibition of Na+,K+-ATPase as compared to the wild type. The increase in abundance of the Na' pump is associated with a 4-fold increase in abundance of the mRNA for the al subunit of the Na+,K+-ATPase. We could not detect mRNA for a2 or a3 or for the .8 subunits. The increase in abundance of the A system and Na+,K+-ATPase is associated with a negligible increase in intracellular Na+ concentration. We propose that the increase in the abundance of the A system and the Na',K+-ATPase is the result of a mutation in regulatory gene RI that controls the A system and the Na',K+-ATPase and is not due to a primary effect of a possible initial increase in Na+ concentration.A study of a constitutive mutant of CHO-K1 cells, alar4, which has increased abundance of the membrane-bound transporter for the A system of amino acid transport (1-3), has revealed an interesting relationship between this system

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“…The serum concentration of the growth medium affects the density of sodium pumps (Aiton & Lamb, 1984), so we examined the effects of growth in ouabain at different serum concentrations. Over 48h, raising serum from 0.1% to 20% increased pump density by 30% but 10nm ouabain produced the same block (66%) at both serum concentrations, with no interaction (by ANOVA).…”

Section: Effect Of Serum and The Potassium Concentrationmentioning

confidence: 99%

Discrepancy between the short and long term effects of ouabain on the sodium pumps of human cells grown in culture

Griffiths

Ogden

Cormack

et al. 1991

British J Pharmacology

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1 Human cells (HeLa) were cultured for periods up to 48 h in growth medium in the absence or presence of a range of concentrations of cardiac glycosides. In some experiments the potassium concentration of the medium was varied between 0.3 mm and the usual 5 mm. 2 For periods up to 2 h in ouabain the association and dissociation rate constants were measured and the equilibrium binding constant (KD) calculated; the apparent equilibrium binding constant (K'D) was measured after 1-2 days growth in ouabain.3 Ouabain had a K' after 2 days of 2-6 nm in 5 mM K' growth medium, a 4 fold greater blocking effect on sodium pumps after 2 days than expected from the association and dissociation rate constants measured in untreated or previously ouabain-treated cells. 4 This effect was: (a) approximately the same over a range of external potassium concentrations from 0.3 to 5 mm, although the absolute effect of ouabain over this range of potassium was much different; (b) probably not due to different isoforms of pumps in cells grown in ouabain compared to untreated cells; (c) apparently not a consequence of internalisation of pump-glycoside complexes. 5 We conclude that ouabain has only a limited access to sodium pumps in whole cells; this could be because sodium pumps cycle continuously through an inaccessible region of the plasma membrane. This effect needs to be considered both in the assessment of the magnitude of the long term effects of cardiac glycosides on cells, and in the measurement of the glycoside affinities of various isoforms of the pump.

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EFFECT OF THE SERUM CONCENTRATION OF THE GROWTH MEDIUM ON THE SODIUM PUMP SITE DENSITY OF CULTURED HeLa CELLS

Cited by 11 publications

References 35 publications

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

Discrepancy between the short and long term effects of ouabain on the sodium pumps of human cells grown in culture

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