Uptake Of 42K Research Articles

Insulin-like growth factor I stimulates active Na(+)-K+ transport in rat soleus muscle

The functional homology between insulin and insulin-like growth factor I (IGF-I) comprises effects on growth and glucose metabolism. Because insulin stimulates the Na(+)-K+ pump, IGF-I might exert a similar effect. We show here that IGF-I increases 42K and 86Rb uptake and the efflux of 22Na in isolated rat soleus muscle. This leads to a significant decrease (21-55%, P < 0.001) in intracellular Na+ and a small increase in intracellular K+. In extensor digitorum longus (EDL) muscle, similar effects were observed. The stimulation of K+ uptake and the reduction in intracellular Na+ in the soleus were blocked by ouabain, indicating that they reflect an acute stimulation of active Na(+)-K+ transport. This conclusion was further supported by the observation that the [3H]ouabain binding rate was significantly increased by IGF-I. IGF-I increased ouabain-suppressible 42K or 86Rb uptake by 56 and 54%, respectively. The effects of IGF-I and epinephrine on ouabain-suppressible 86Rb influx in rat soleus were additive, whereas the effects of insulin and IGF-I were similar and nonadditive. The effects of IGF-I were seen down to a concentration of 10(-8) M, which is unlikely to stimulate the insulin receptor, and it is therefore plausible that IGF-I exerts its effect on Na(+)-K+ transport through its own receptor. IGF-I may play a role in the maintenance of muscle Na+ and K+ contents also in vivo, especially in patients treated with IGF-I.

86Rb is not a reliable tracer for potassium in skeletal muscle

For technical reasons, 86Rb is frequently preferred to 42K as a tracer for K+. Systematic comparisons of the two isotopes, however, are rarely done. In this paper we compare the transport of 42K and 86Rb in rat and mouse soleus muscle and in rat erythrocytes. Ouabain-suppressible K+ uptake in rat soleus was the same whether measured with 42K or 86Rb, both when stimulated by insulin, salbutamol and calcitonin-gene-related peptide (CGRP), and when inhibited by graded concentrations of ouabain. Control experiments with rat erythrocytes, where Na(+)-K(+)-Cl- co-transport has earlier been demonstrated, showed closely similar inhibitory effects of bumetanide on 42K and 86Rb uptake. In contrast, bumetanide produced no significant change in 42K uptake of rat and mouse soleus muscle, but clearly inhibited 86Rb uptake at concentrations down to 10(-7) M (P < 0.001). Whereas the addition of 150 mM NaCl had no effect on 42K uptake in rat soleus, 86Rb uptake, and in particular the bumetanide-suppressible component, was markedly increased by this addition. The inhibitory effect of bumetanide on 86Rb uptake gives rise to the false impression that skeletal muscle contains a NaKCl2 co-transport system. Efflux studies showed that the fractional loss of 42K from rat soleus muscle is 2.3 times larger than that of 86Rb. Salbutamol and CGRP increased 86Rb efflux, but inhibited 42K efflux. This implies that for studies of K+ efflux and bumetanide-sensitive K+ transport, 86Rb is not even an acceptable tracer for the detection of qualitative changes. Control experiments with 42K are essential in any characterization of unknown K+ transport processes.

Effects of extremely-low-frequency electromagnetic fields on ion transport in several mammalian cells.

We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca2+, Na+, K+, and H+) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca2+ concentration ([Ca2+]i), the intracellular pH (pHi), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100-2000 microT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20-1200 microT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12-32% increase of the uptake of 42K within a window at 14.5-15.5 Hz and 100-200 microT (rms), which was found in U937 and Ehrlich cells but not in the other cell types.

Effects of fasting, refeeding, and fasting with T 3 administration on Na-K,ATPase in rat skeletal muscle

It is known that Na-K,adenosine triphosphatase (ATPase) in cell membranes represents an important consumer of cellular energy, eg, adenosine triphosphate (ATP), and that the concentration and activity of this enzyme change in a dose-dependent manner with serum thyroid hormone levels. To examine the hypothesis that low triiodothyronine (T 3) syndrome represents a cellular adaptation in generalized severe illnesses that saves tissue energy expenditure, we measured the muscle Na-K,ATPase concentration and its activity in rats that led to low T 3 syndrome induced by fasting. The Na-K,ATPase concentration was measured by 3H-ouabain binding to soleus muscle, and its activity was measured by 42K uptake in the contralateral soleus muscle. The effects of refeeding or T 3 administration on Na-K,ATPase in soleus muscle in fasted rats were also examined. Na-K,ATPase concentration and activity were both increased in hyperthyroid rats and decreased in hypothyroid rats. In the fasting state, they were decreased to as low as the levels seen in hypothyroidism. Furthermore, with fasting + refeeding or fasting + T 3 administration, Na-K,ATPase in soleus muscle returned to the normal level. These results suggest that tissue energy expenditure, as assessed by Na-K,ATPase, in skeletal muscles of fasted rats with low T 3 syndrome is actually decreased to levels seen in hypothyroidism, due at least partly to the decrease in serum T 3 concentrations, and that there exist some adaptation mechanisms in the peripheral tissues for the accomodation of energy metabolism in the body through decreased thyroxine (T 4) to T 3 conversion.

Foliar fertilization of 42KCl in Phaseolus vulgaris L.

Distribution and uptake rate of42KCl solution when applied as foliar fertilizer were measured.42 KCl tracer solution was prepared by the milking method from42Ar-42K generator. The total uptake ratio of 42K was about 20% in 24 h. To enhance the uptake rate and amount, leaf moisturing method was effective, which raised the uptake ratio three times higher. When the solution was applied at the back side of the leaf, the absorbed amount was 10 to 20% higher than that amount applied at the surface side of the leaf. The amount of 42K uptake measured in this report suggests that the foliar fertilization of K can be the econo-mical way of fertilization in Brazil.

Alterations in cation homeostasis in cultured chick ventricular cells during and after recovery from adenosine triphosphate depletion.

Alterations in cation homeostasis during and after recovery from myocardial ischemia may account for some of the reversible and irreversible components of myocardial cell injury. To investigate possible mechanisms involved, we exposed cultured layers of spontaneously contracting chick embryo ventricular cells to media containing 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG), and zero glucose for up to 6 h, and then allowed cultured cells to recover in serum-free culture medium for 24 h. Changes in Na, K, and Ca contents, 42K uptake and efflux, ATP content, cell water content, and lactate dehydrogenase (LDH) release were measured, and compared with changes produced by exposure to 10(-3) M ouabain and severe hypoxia. Exposure to CN and 2-DG caused marked increase in cell Na (sevenfold) and Ca (fivefold) contents, and a decrease in K content (one-fifth normal), coincident with ATP depletion to one-tenth normal levels. This produced only slight cell injury, evidenced by increased LDH release. Recovery for 24 h resulted in return to near normal values (expressed in nanomoles per milligram of protein) of Na, Ca, and ATP contents. However, there was failure of cell K content to return to normal, associated with a persistent reduced net uptake of 42K, and an increase in the rate of 42K efflux. These abnormalities in K homeostasis were associated with a decrease in cell volume and water content per milligram of protein. More marked ATP depletion (to 1/100 normal values) was produced by hypoxia plus 2-DG and zero glucose, and was associated with much more severe cell injury manifested by LDH loss. Ouabain exposure resulted in a much greater Ca gain (20-30-fold), relative to increase in Na content, than did either CN and 2-DG or hypoxia; and ouabain effects were not reversible (after a 15-fold or greater increase in Ca content was produced) and were associated with significant LDH release. We conclude that these cells are resistant to cell injury caused by moderately severe Ca overload and ATP depletion produced by exposure to CN and 2-DG. However, metabolic inhibition of ATP production produces persistent abnormalities in K homeostasis, associated with functional abnormalities.

Na-K pump site density and ouabain binding affinity in cultured chick heart cells

The possible existence of multiple [3H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [3H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s-1 [corrected]. Maximal specific [3H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [3H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [3H]ouabain and 125I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [3H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells.

The role of extracellular sodium on heart muscle energetics.

A study has been made of changing external sodium concentration [Na]e, over the range 75 to 200 mmol X l-1, on contractile parameters and heat production in isolated, arterially perfused, interventricular rabbit septa.- The observed changes in maximum rate of contraction with [Na]e, either in the presence of a constant external Ca concentration [Ca]e or in the presence of a constant [Na]e2/[Ca]e ratio, paralleled those observed for tension development (T). On the other hand the maximal rate of relaxation (-Tmax) and the ratio -Tmax/T increased. While the ratio between active heat production and developed tension remained unaltered (0.111 +/- 0.003 mJ X mN-1 X g-1 dry weight), resting heat production increased with [Na]e2 with a slope of 95 +/- 18 mW X g-1 X mol-2 X l2. Under resting conditions, a decrease in [Na]e of 50 mmol X l-1 induced a fall in 42K uptake of about 16 nmol X s-1 X g-1 without changes in 42K efflux, suggesting that such an intervention depresses K influx. If the depressed K influx, induced by a decrease in [Na]e of 50 mmol X l-1, is associated with a decrease in Na-K pump activity, a fall in resting heat production of about 0.64 mW X g-1 would be expected. This represent 56% of the calculated change in the resting heat production, 1.14 +/- 0.22 mW X g-1 (mean +/- one confidence interval), suggesting that some process in addition to a depressed Na-K pump activity may be altered by changes in [Na]e.

Effects of Age on Cell Size and Ion Uptake in Canine Cortical Bone

In cortical bone from dogs of various ages, the uptakes of two bone-seeking isotopes, 85Sr and 99mTc-methylene diphosphonate (MDP), were compared with that of the intracellular cation 42K by using the volume of distribution (VD) technique. The rate of bone remodeling and the osteocyte volume were estimated by morphometric techniques. The percentage cell volume was related to the VD of 42K (r = 0.83; P less than 0.001) and decreased with increasing age. The decline in the VD of 85Sr and 99mTc-MDP with aging was related to the decrease in bone formation (r = 0.90; P less than 0.001). On a logarithmic plot, the cell volume and the VD of 42K had a linear relationship with the rate of new bone formation, the VD of 85Sr, and the VD of 99mTc-MDP. Our results demonstrate that, with increasing age in dogs, the bone formation rate and cell size decrease, as does the uptake of 42K, 99mTc-MDP, and 85Sr.

Adrenaline causes potassium influx in skeletal muscle and potassium efflux in cardiac muscle in rats: The role of NA/K ATPase

Previous in vitro evidence suggest that adrenaline causes K influx in skeletal muscle by stimulating a ouabain sensitive Na/K ATPase membrane pump. However in rabbits, adrenaline induced hypokalaemia was not significantly altered by pretreatment with digoxin (50μg/kg). Rats were infused with adrenaline or saline after being given a tracer dose of 42KCl. Adrenaline caused a highly significant uptake of 42K in skeletal muscle and a decrease in 42K uptake in ventricle. Rats were also studied after receiving a high dose of digoxin (1.4 mg/kg) which by itself produced a significant increase in plasma K, a decrease in plasma Na and a decreased uptake of 42K in ventricle and lung. These results suggest that adequate widespread Na/K ATPase inhibition had been achieved by this dose of digoxin but despite this, adrenaline still caused hypokalaemia and also still caused significant 42K tissue uptake by skeletal muscle. These results suggest that adrenaline causes K influx by skeletal muscle and K efflux by cardiac tissue. Furthermore the former mechanism was not inhibited by pretreatment with digoxin.

Potassium loss from rabbit myocardium during hypoxia: Evidence for passive efflux linked to anion extrusion

To determine the effects of permeant and impermeant anions and of osmolarity on potassium (K+) exchange, the net uptake and efflux of 42K+ were recorded in the isolated arterially perfused rabbit septum. Perfusion with solution made hyperosmolar by adding NaCl (30 mM) or sucrose (60 mM) caused similar increases of 42K+ uptake which were reversible on returning to the control solution. Washout experiments showed that the loss of K+ on returning to the control perfusate was due to a decreased influx probably mediated by inhibition of the sodium pump. The effects of anions were studied by replacing chloride in the control solution with the inert and impermeant substitute isethionate (114 mM) or by loading the myocardium with sodium dimethyloxazeolidinedione (NaDMO, 30 mM) under isosmotic condition and switching to a perfusate containing sodium isethionate (30 mM). In both these conditions a reduction of 42K content could be detected and was attributable to an increased efflux. During hypoxic substrate free perfusion K+ loss was due to an increased efflux with no evidence for altered influx of potassium. The extrusion of accumulated anions from the myocardium could be the major determinant of the early potassium loss during hypoxia and ischaemia.

Hyperthermia and cytotoxic drugs. Possible use of lanthanum as a potentiator of hyperthermia.

Recent studies indicate that differences in membrane fluidity may account for differences of thermal sensitivity. This possibility was studied by using lanthanum, a trivalent cation which is known to displace calcium in a number of biological systems, to modify the structural framework of cell membranes and consequently their biological properties. With Ehrlich ascites cells trypan blue exclusion uptake of 86Rb, 42K and 45Ca, indicate an increase of plasma cell permeability by La3+. The reduction of 86Rb and 42K uptake by tumour cells with La3+ appears to be independent of temperature. The increase of 45Ca2+ influx in the presence of lanthanum plus hyperthermia seems related to an important loss of cell viability. The enhancement of hyperthermia lethality by concentrations of lanthanum over 0.5 mM after 2 h at 44 degrees C has been demonstrated using HeLa S3 cells using a standard cloning technique. In vivo experiments have been performed on C3H mice bearing rhabdomyosarcoma using ultrasound heating at 44-46 degrees C. The results show a remarkable inhibition of tumour growth and a significant increase of the survival time after only one hyperthermia session of 30 min combined with one intratumoural injection of 1 mM lanthanum chloride.

Decreased sodium-potassium pump activity in isolated hypertrophied feline ventricular myocytes

The activity of the Na-K pump was assessed in normal and hypertrophied isolated feline myocytes by measuring ouabain-sensitive 42- K uptake. Right ventricular hypertrophy was produced in feline myocardium by placing a constricting band around the pulmonary artery of adult cats. High yields of calcium tolerant myocytes were isolated from the right and left ventricle of banded and sham operated animals. Intracellular sodium (Na) and potassium (K) concentrations (mM) were not significantly different (P > 0.5) in normal (Na: 13.2 ; K: 133.4) and hypertrophied (Na: 12.3 ; K: 127.5) myocytes. Morphometric analysis demonstrated a 26% increase in width and a 42% increase in volume of hypertrophied myocytes, however, the sarcomere length (1.9 μ) was not different in both cell types. The rate constant (k, min −1) describing 42-K uptake and the calculated total K influx (I, pmol/cm 2/sec) were not significantly different (P > 0.5) in normal (k=0.059 ; I=15.9) and hypertrophied (k=0.062; I=15.3) cells. Ouabain-sensitive (active) K influx, a measure of Na-K pump activity, was maximally inhibited at 10 −4M ouabain in both cell types. At this concentration, ouabain-sensitive K uptake was decreased 23.5% in hypertrophied myocytes compared to control. The decrease in active K influx may be due to a decrease in the activity of the Na-K ATPase and/or to a reduction in the passive movement of sodium and potassium down their electrochemical gradients.

Differences in cation transport properties of primary astrocyte cultures from mouse and rat brain

42K and 22Na contents and unidirectional fluxes, as well as net accumulation of 42K in response to elevated extracellular K +, were investigated in primary cultures of astrocytes prepared from neonatal rat and mouse brain. The major difference between both species affected the unidirectional K + influx which was up to 75 times higher in mouse as compared to rat cultures. The flux rates in mouse astrocytes were doubled by measuring uptake in salt solution instead of growth medium, while 42K influx in rat astrocytes was unaffected by such treatment. 22Na transport was very similar in astrocytes from both species. The length of culture period and treatment with DBcAMP (2′,3′-dibutyryl cyclic adenyl monophosphate) modified K + transport but not Na + transport. Both types of cultures showed the same accumulation of 42K in response to raised medium K +. Amiloride inhibited 42K influx by 41% and 13% in mouse and rat cultures, respectively. In contrast, furosemide inhibited 42K uptake in rat astrocytes cultures by 50% but had no effect on mouse astrocyte cultures. 50 μM barium chloride markedly inhibited 42K uptake in mouse cultures by 96% (or 1491 nmol·mg −1·min −1), but inhibited 42K uptake in rat cultures by only 23% (or 9 nmol·mg −1·min −1). Ouabain was similarly effective in both types of astrocyte cultures. We conclude that Na + transport as well as net K + accumulation and Cl − transport (based on previous studies) properties are reasonably stable and reproduced in primary cultures from both mouse and rat brain. However, unidirectional K + influx showed a large species difference and also a larger variation due to differences in culturing conditions. There were also differences in the sensitivity to some inhibitors. Possible causes could be differences in the number or characteristics of K + channels as well as other specific K + transport systems.

Alterations to potassium fluxes in the heart during chronic dietary potassium depletion

Dietary K+ depletion in rabbits causes a loss of cellular K+ from skeletal muscle but not from cardiac muscle, and it has been suggested that this is due to a specific protective mechanism in the heart. Net uptake and efflux of 42K was investigated in the interventricular septum of the rabbit. Acute reductions in extracellular [K+] caused a loss of K+ from tissue of both normal and K+ depleted animals. In the K+ depleted group the relationship between [K+]e and net uptake was altered, so that the maximum rate of uptake was increased, and this was obtained at a lower [K+]e. There were no significant changes in K+ efflux. These results would be consistent with an adaptive change to the cardiac cell Na+ pump during chronic K+ depletion.

Sodium Exclusion from the Shoots by Roots ofZea mays(cv. LG 11) and its Breakdown with Oxygen Deficiency

The effects of sodium chloride salinity and root oxygen deficiency (anoxia) were studied in 11-12 d old maize plants (Zea mays L. cv. LG 11) in nutrient solution culture. Transport of 22Na by the roots to the shoot in 24 h was markedly increased by anoxia when the external concentration of NaCl was in the range 01-10-9 mol m~3. Anoxia severely inhibited uptake of 42K by roots and its transport to the shoot, so that the ratio of Na+/K+ moving into the shoot was increased by a factor of approximately 10. When the external concentration of NaCl was increased to 40-0 mol m~3, the roots showed much less ability to exclude Na+ under aerobic conditions, and anoxia caused no further increase in the movement of Na+ to the shoot. It is concluded that at the higher concentration the ability of the roots to exclude Na+, presumably through an active mechanism in the xylem parenchyma cells or in the root cortex and transporting Na+ to the outer solution, is saturated by excessive inward diffusion of Na+. The ratio of Na+/K+ transported to the shoot increased by a factor of 600 when the concentration of NaCl was increased from 2-4 mol m ~ 3 to 40 mol m ~ 3 and roots were made anoxic. Such imbalances in the supply of cations to the shoot, particularly when roots are oxygen-deficient, may contribute to salinity damage.

Active ion transport in the renal proximal tubule. I. Transport and metabolic studies.

Various aspects of the interrelationship between ion transport and cellular metabolism were investigated using a suspension of rabbit cortical tubules that were mainly proximal in nature. Using the intact tubules, the compartmentation of K within the renal cell was studied by performing 42K uptake studies. The oxygen consumption (QO2) of the tubules was measured under similar conditions, as well as when the Na pump was stimulated by increasing Na+ entry with nystatin. In addition, the state 3 rate of respiration was measured when the mitochondria of digitonin-permeabilized tubules were stimulated by ADP. At 37 and 25 degrees C, a single-compartmental uptake of 42K was observed, which suggests that extracellular K+ communicates with a single compartment within the renal cell. Between 37 and 15 degrees C, the ouabain-sensitive QO2 and the initial 42K uptake rate were parallel in an Arrhenius-type plot, which indicated that active ion transport and oxidative phosphorylation remain tightly coupled within this temperature range. At all temperatures between 37 and 15 degrees C, nystatin stimulated the QO2, which demonstrates that the entry of Na+ into the renal cells was rate limiting for active Na+ transport throughout this temperature range. Between 37 and 20 degrees C, the nystatin-stimulated QO2 was nearly equal to the state 3 rate of respiration, which suggests that active ion transport may be limited by ATP availability under these conditions. At 15 degrees C, nystatin addition stimulated the QO2 well below the state 3 respiratory rate.

Myocardial metabolic inhibition and membrane potential, contraction, and potassium uptake.

Interruption of synthesis of ATP during hypoxia or ischemia can produce membrane depolarization that may be related to inhibition of Na+-K+-ATPase. To examine this hypothesis, the effects of exposure of cultured chick embryo ventricular cells to 1 mM cyanide (CN), to 20 mM 2-deoxy-D-glucose (2-DG), to CN + 2-DG, and to ouabain (10(-3) M) on contraction, membrane potential, and 42K uptake were determined. CN produced moderate membrane depolarization and electromechanical uncoupling within 2 min. 2-DG caused marked membrane depolarization with a transient negative inotropic effect. Exposure to CN + 2-DG produced marked depolarization (-38 mV) and mechanical arrest of the cells in a relaxed state. Ouabain produced marked depolarization (-39 mV) and contracture of the cells. Uptake of 42K was inhibited by 10(-3) M ouabain within seconds. However, CN + 2-DG produced no inhibition of 42K uptake within the first 2 min of exposure, and inhibition of the Na pump by CN + 2-DG required 30 min to develop fully. Exposure to CN alone produced no inhibition of 42K uptake, whereas moderate inhibition was produced by 2-DG alone even when substrate for oxidative phosphorylation was provided. We conclude that the acute effects of inhibition of glycolysis and oxidative phosphorylation on membrane potential and contraction in these cells are not due to inhibition of the Na pump and that during partial metabolic inhibition active univalent cation transport in these cells is relatively dependent on ATP derived from glycolysis, whereas contraction is more dependent on ATP supplied by oxidative phosphorylation.

O6-Ethylguanine is not the only ethylated adduct responsible for the induction of mutations in Syrian hamster embryo (SHE) cells

Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5–6 mM K+, or ≥ 0.1 mM with 0.5 mM K+, inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUAR) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUAR L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUAR cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUAR clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUAR cells in a wild-type population can be substantially increased, to approximately 10−4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5–6 × 10−8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUAR and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

Cell volume change of Escherichia coli under stringent control apparent increase of K + in rel− cells

With several pairs of rel + and rel − strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K + and the rate of uptake of 42K + were investigated. In the early phase of the experiments, the intracellular concentration of K + was estimated by the conventional method in which the cell volume per A 660 value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K + concentration of rel + cells was kept almost constant, while that of rel − cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A 660 changed only slightly in CP78 ( rel +) cells, while it increased markedly in CP79 ( rel −) cells after the exposure to amino acid starvation. Reestimation of the K + concentrations based on the estimated respective values of cell volumes per A 660 revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel + cells was arrested upon amino acid starvation whereas that in the rel − cells was not. The 42K + uptake by the rel + cells was depressed upon amino acid starvation, whereas that by the rel − cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K + concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A 660 of the culture, was pointed out.