Influence of physical factors on the growth of insect cells in vitro

Summary The tolerances of a cell line (IMC-HZ-1) from a moth,Heliothis zea, for the monovalent cations Na⁺ and K⁺ were defined. Cells shifted to media containing more than 70mm of K⁺ showed decreased growth rates. No evidence was obtained for Na⁺ toxicity. The osmotic pressure tolerances were influenced by the K⁺ concentration of the medium. The richer the medium was in K⁺, the narrower was the spectrum of osmotic pressure tolerance. Once the limit of K⁺ tolerance was exceeded, the rate of decline of growth was linear with respect to further increases in K⁺. This rate of decline was independent of osmotic pressure. The initial responses of cells during one subculture (2 to 4 population doublings) in media differing from the standard medium (used to maintain the cell line) were not reliable indicators of the growth potential of the cells. Continued subculture in such media resulted in an upward trend in population growth rates in most cases. This investigation was supported by U. S. Public Health Service Research Grant no. AI 09914 from the National Institute of Allergy and Infectious Diseases. This is Paper no. 8637, Scientific Journal Series, Minnesota Agricultural Experiment Station. The material is part of the dissertation of T. J. K. presented for the Ph.D. degree at the University of Minnesota.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Some properties of an unidentified halophile: growth characteristics, internal salt concentration, and morphology.

An unidentified halophile isolated from plates of a complex agar medium containing 4.25 M NaCl showed optimum growth in broths containing 0.5-1.0 M NaCl but exhibited a wide range of growth from 0.045-4.5 M. The organism can be classified as a facultative halophile with wide salt tolerance. Logarithmic phase cells grown in media containing 0.5 M NaCl were rod-shaped in long chains which changed to smaller, single, or paired cells in stationary growth. The internal Na+ and K+ concentrations were 0.05 M and 0.34 M for logarithmic phase cells and 0.29 and 0.32 M for stationary phase cells. In 4.3 M NaCl media the cells were rod-shaped throughout the growth cycle, occurring primarily in pairs. The internal Na+ K" concentrations in cells in logarithmic phase growth were 0.62 M and 0.58 M while in stationary phase growth these values were 1.01 M and 0.66 M respectively. In contrast, logarithmic phase cells of the extreme halophile Halobacterium cutirubrum had internal Na+ and K+ concentrations of 0.80 M and 5.32 M when grown in 3.3 M NaCl. The internal Na+ and K+ concentrations, therefore, in the unidentified halophile do not resemble those found in H. cutirubrum but are much closer to those present in Escherichia coli.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Isoosmotic regulation of cotton and peanut at saline concentrations of k and na

Peanut (Arachis hypogaea L.) and cotton (Gossypium hirsutum) plants were grown for 4 weeks in saline, isoosmotic rooting substrates with different proportions of K and Na. Isoosmotic media did not affect growth (except at the highest external K concentrations) or estimates of intracellular osmotic pressure in expanding leaves (i.e. osmotic pressure of leaf sap and intracellular osmotic pressure as calculated from pressure-volume curves). In expanded leaves, an increase in the proportion of external K increased sap osmotic pressure. The sum of [K+Na+Cl] in the sap of expanding and expanded leaves accounted for the effect of isoosmotic media on the concentration of osmolytes with high electrical conductance, so the difference between sap osmotic pressure and [K+Na+Cl] accounted for the concetration of osmolytes with low conductance. In expanding leaves, an increase in the proportion of external K increased [K+Na+Cl] and decreased the concentration of osmolytes with low conductance. In expanded leaves, an increase in the proportion of external K increased [K+Na+Cl] to approximately the same extent as sap osmotic pressure. Isoosmotic regulation was apparent in expanding leaves but not evident in expanded leaves. This suggests a turgor homeostat which can influence the concentration of organic solutes in expanding leaves but cannot control the import of inorganic solutes from a rooting medium nor the total production of organic solutes in plants with a low sink:source ratio.     

Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions.

To assess the physiological function of Ca(2+)-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.     

K+-induced swelling of the dogfish shark (Squalus acanthias) rectal gland cells is associated with changes of the cytoskeleton

Dogfish shark (Squalus acanthias) rectal gland cells swell massively when incubated in elasmobranch media in which Na+ was equivalently replaced by K+; this swelling was abolished when the impermeant gluconate replaced Cl-, while the cell depolarization was comparable in both media. The K+-effect was associated with (a) an increase of the steady-state 42K (and 86Rb) efflux (particularly of the rate constant of the fast cellular efflux component) and a rearrangement of the respective cellular pools of K+; (b) an alteration of cell morphology and the pattern of the F-actin staining along the basolateral cell membrane as revealed with fluorescent analogs of phallacidin. These changes were independent of cell volume, being identical in KCl and K-gluconate media. The observations were specific for K+ (and Rb+): replacement of media Na+ by Li+ (which is not actively extruded by the cells), or the presence of ouabain, produced only minor swelling without affecting cell morphology and F-acting distribution. The results are consistent with the following view: as opposed to Na+ or Li+ media, the K+-induced changes of the cortical F-actin component of the cytoskeleton permit the observed massive cell swelling due to the osmotic contribution of intracellular impermeant anion(s).     

Changes in intracellular K+ and Na+ ion concentrations during cell growth and differentiation

We compared intracellular K+ and Na+ ion concentrations during cell growth and differentiation of a mouse myeloid leukemia M1 cell line. Cells undergoing mitosis had higher K+ concentrations than quiescent cells. Treatment with a K+ channel blocker and furosemide enhanced cell growth and produced a slight increase in the intracellular K+ concentration. Treatment with reagents that reduced the intracellular K+ concentration stopped cell growth. Induction of differentiation in this cell line produced a decrease in the K+ concentration, which always was accompanied by an increase in the Na+ concentration. Treatment with ouabain, which decreased the intracellular K+ concentration, did not, however, induce differentiation in the M1 cell line. The data suggest that cell growth and differentiation in the M1 cells are accompanied by changes in the intracellular K+ and Na+ concentrations but that the changes in the contents of these monovalent cations do not necessarily induce differentiation in this cell line.     

Trimethylamine oxide and the maintenance of volume of dogfish shark rectal gland cells

Determinants of the steady-state vol of the dogfish shark (Squalus acanthias) rectal gland cells were studied. The cellular levels of trimethylamine oxide (TMAO) in fresh tissue and slices incubated aerobically 60 min in standard (TMAO-free) elasmobranch saline were close to those in the plasma (71 +/- 5 mM S.E.M.); therefore, under these conditions, the cell membrane appears to be impermeable to this solute. However, depolarization of the cells in high-K+ media produced a rapid loss of TMAO. Thus, TMAO is a major, effectively impermeant solute in the rectal gland cells. The osmolarity of cell solutes in tissue water (fresh and incubated slices) did not differ significantly from values in the plasma or incubation medium, demonstrating the absence of an osmotic pressure gradient across the cell membrane. An analysis of a simple model of cell solutes under steady-state conditions shows that the presence of an (effectively) impermeant osmolyte decreases the cellular concentration of bulk cations. The analysis is consistent with available observations on the distribution of cell Na+ and K+ in tissues containing high concentrations of (nitrogeneous) osmolytes. One simplifying assumption of the model, i.e., identity (or closeness) of the respective reflection coefficients sigma for Na+ and K+ passage through the cell membranes could not be verified. Compared to available data on the steady-state cellular fluxes of 42K+ in slices of the rectal gland, the uptake of 22Na+ by the tissue was slow (the derived rate constant k' = 0.017 min-1, i.e., about one tenth of that for K+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of TRH and somatostatin on releases of prolactin and growth hormone in vitro by the pituitary of Poecilia latipinna

Summary Pituitary glands from a teleost fish were incubated in the presence of the synthetic hypophysiotropic peptides, thyrotrophin-releasing hormone and somatostatin, in two media of different osmotic pressure.The effects on prolactin and growth hormone cells were detected by electron-microscopic morphometry with the aid of an image analyser. Thyrotrophin-releasing hormone caused changes in prolactin cell ultrastructure consistent with stimulated hormone release and, in the low osmotic pressure medium, appeared to increase synthetic activity. There was no effect on growth hormone cells. After somatostatin treatment, both synthesis and release in prolactin cells appeared to be inhibited, and there was an obvious inhibition of synthesis and release in growth hormone cells. The response of both cell types to somatostatin did not appear to be dependent on the osmotic pressure of the medium.     

L-929 cells under hyperosmotic conditions. Water, Na+, and K+

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using 14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of in-folding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.     

The effect of the osmotic pressure of the culture medium on cells of Francisella tularensis vaccinal strain 15

The dependence of the growth rate of F. tularensis on the osmotic properties of the medium can be presented as a curve with the maximum in the area of 500-600 mOsm. Under these circumstances the intracellular osmotic pressure exceeds the extracellular one by 50-100 mOsm. With the rise of the osmotic pressure in the medium the increase of the concentration of K+ in the cells occurs. The energy-dependent accumulation of K+ in the cells at rest is activated by the rise of the osmotic pressure in the medium. F. tularensis are probably capable of osmoregulation, ensured by the energy-dependent osmosensitive K(+)-transporting system.     

Effect of osmotic pressure,Na+/K+ ratio and medium concentration on the enzyme activity and growth of L cells in suspension culture

Summary The effect of changes in osmotic pressure and in the Na⁺/K⁺ ratio on the doubling time, maximum cell population, enzyme activity, and isoenzyme distribution pattern in suspension cultures of L cells was determined. The growth of viable cells is relatively flat over a rather wide range of osmotic pressures (220 to 440 mOsm per kg). The presence of extra salt or sucrose beyond that needed to reach the minimum osmotic pressure (220) is detrimental to cell growth as reflected by a delay in the onset of logarithmic growth, a slower growth rate, a decreased maximum population, and accelerated death phase. Excessive K⁺ ion is toxic, but the level at which it is toxic interacts with osmotic pressure of the medium. Enzyme activity and isoenzyme distribution patterns for those enzymes studied did not vary as a function of osmotic pressure, ionic ratios, or medium concentration.     

In vivo and in vitro research on the biological effects of deuterium-depleted water: 1. Influence of deuterium-depleted water on cultured cell growth.

Deuterium depleted-water (DDW) is a new available tool for decreasing deuterium concentration in the environment of cells in culture. Several types of established cell lines, both normal and neoplastic were grown in culture media dissolved with DDW and compared with the same strains, in the same amounts, grown in media dissolved with normal distilled water. Naive mice splenocytes were grown, under stimulation with proliferation triggers, like bacterial lipopolysaccharide (LPS) and Concanavalin A (ConA) in the same conditions. The growth and proliferation were estimated using the MTT assay. Both established cell types and explanted splenocytes in the DDW-media had a significantly higher growth rate than cell cultured in normal media. In an attempt to identify the membrane mechanisms involved in the growth stimulation by DDW, the membrane proton transporters Na+/H+ antiporter and H+/K+ATP-ase were inhibited with their selective blockers amiloride and respectively lansoprazole. The results, however incomplete, point towards a lack of involvement of the Na+/H+ antiporter and a possible implication of the H+/K+ATP-ase.     

Effect of volume changes on ouabain-insensitive net outward cation movements in human red cells

Summary The effect of cell volume changes in human red cells on ouabain-insensitive net outward cation movements through 1) the Na–K and Li–K cotransport, 2) the Li–Na counter-transport system and 3) the furosemide-insensitive Na, K and Li pathway was studied. Cell volume was altered by changing a) the internal cation content (isosmotic method) or b) the external osmolarity of the medium (osmotic method). Na–K and Li–K cotransport were measured as the furosemide-sensitive Na or Li and K efflux into (Na, Li and K)-free (Mg-sucrose replacement) medium from cells loaded to contain approximately equal concentrations of Na and K, or a constant K/Li concentration ratio of 91, respectively. Li–Na countertransport was assayed as the Na-stimulated Li efflux from Li-loaded cells and net furosemide-insensitive outfluxes in (Na, Li and K)-free media containing 1mm furosemide. Swelling of cells by the isosmotic, but not by the osmotic method reduced furosemide-sensitive Na and Li but not K efflux by 80 and 86%, respectively. Changes in cell volume by both methods had no effect on Li–Na countertransport. The effects of cell volume changes were measured on the rate constants of ouabain- and furosemide-insensitive cation fluxes and were found to be complex. Isosmotic shrinkage more than doubled the rate constants of Na and Li efflux but did not affect that of K efflux. Osmotic shrinkage increased the K efflux rate constant by 50% only in cells loaded for countertransport. Isosmotic cell swelling specifically increased the K⁺ efflux rate constants both in cells loaded for cotransport and countertransport assays while no effect was observed in cells swollen by the osmotic method. Thus, the three transport pathways responded differently to changes in cell volume, and, furthermore, responses were different depending on the method of changing cell water content.     

Influence of temperature on ionic sparing effect and cell-associated cations in the moderate halophile, Micrococcus varians var. halophilus

Cells of the moderately halophilic Micrococcus varians var. halophilus grew well in a chemically defined medium containing 1 to 3 M NaCl and 0.0103 M K+. The requirement for NaCl could be partially replaced by K+,:Li+ and Cs+. The efficiency of the sparing effect of these cations for NaCl was in order of K+ GReater than Li+ greater than Cs+. Increase in growth temperature was found to enchance the sparing effect of Li+ and Cs+ but not that of K+. Over the range of NaCl concentrations in which the cells grew well, cell-Na+ concentrations were similar to the medium NaCl concentrations while cellK+ concentrations were several-fold that in the medium. Cell-bound Na+ and K+ concentrations increased proportionally with medium NaCl concentration and growth temperature. The temperature-dependent cation accumulation was more obvious with K+ than Na+. The cell-associated Na+ + K+ concentrations were almost as high as or slightly higher than the external media which contained appropriate levels of NaCl regardless of the growth temperature.     

Osmoregulation of the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat at different salinities.

The intracellular solute composition of the salt-tolerant yeast Debaryomyces hansenii was studied in glucose-limited chemostat cultures at different concentrations of NaCl (4 mM, 0.68 M, and 1.35 M). A strong positive correlation between the total intracellular polyol concentration (glycerol and arabinitol) and medium salinity was demonstrated. The intracellular polyol concentration was sufficient to balance about 75% of the osmotic pressure of the medium in cultures with 0.68 and 1.35 M NaCl. The intracellular concentration of K+ and Na+, which at low external salinity gave a considerable contribution to the intracellular water potential, was only slightly enhanced with raised medium salinity. However, the ratio of intracellular K+ to Na+ decreased; but this decrease was less drastic in the cells than in the surrounding medium, i.e., the cells were able to select for K+ in favor of Na+. The turgor pressure, which was estimated on the basis of intracellular solute concentrations, was 2,200 kPa in cultures with 4 mM NaCl and decreased when the external salinity was raised, resulting in a value of about 500 kPa in cultures with 1.35 M NaCl. The maintenance of a positive turgor pressure at high salinity was mainly due to an increased production and accumulation of glycerol.     

Volume-regulating behavior of human platelets

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidimetry. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCl via volume-activated conductive permeability pathways for K+ and anions, presumably Cl-. In media containing greater than 50 mM KCl, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl-. The K+ pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl- pathway accepted NO-3 and SCN- but not citrate or SO4(2-). In isotonic medium, the permeability of platelets to Cl- appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl- permeability exceeded that of K+. The Cl- conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+/H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.     

Environmental factors for the growth of animal cells

Summary Although cells can grow throughout a wide range of osmotic pressures, any additional salt beyond that required to meet the minimal level exerts an adverse effect, as evidenced by delay in onset of logarithmic growth, slower growth rate, decreased maximal population, and accelerated death. The toxicity of K⁺ is directly related to the osmotic pressure of the culture. Cells appear to ingest most of the nutrients from the medium within the 1st day after being inoculated into it and do not remove significant amounts of nutrients for the next 7 to 8 days of active growth. Dissolved oxygen and medium oxidation-reduction potential (ORP) do not always vary identically and, of the two parameters, ORP is the more critical. If the ORP of the culture is not held constant during growth, enzymatic activity fluctuates during the culture growth cycle. However, if the ORP is held constant, then enzymatic activity and isoenzyme distribution patterns remain constant during the growth cycle. For L cells, the optimum ORP is 100 mv. The ORP of the medium controls the rate and extent of growth of cells. If the ORP is lowered, cells can be inoculated into media and held at 37°C for at least 5 days with no detrimental effect; then, when the ORP is raised, the cells grow as well as if they had just been inoculated into fresh media. By controlling the osmotic pressure, media composition, and environmental parameters, high yields of cells and virus may be consistently obtained. Presented at the Twenty-fifth Annual Meeting of the Tissue Culture Association in the Paul F. Kruse, Jr. Memorial Symposium on Steady-State Culture of Cells.