Effect of low extracellular Ca2+ on growth spreading area,cytoplasmic Ca2+ concentration,and intracellular pH in normal and transformed human fibroblasts

The transformation of certain cells reduces the requirement of extracellular Ca²⁺ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca²⁺ than normal WI-38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca²⁺ medium. Intracellular calcium concentration ([Ca²⁺]_i), of the normal and transformed cells cultured in 10μM and 2 mM Ca²⁺ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca²⁺], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca²⁺],_i of both normal and transformed cells. Although the total decrease in [Ca²⁺]_i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca²⁺ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca²⁺]_i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pH_i) of normal and transformed cells maintained at low and normal Ca²⁺. The low Ca²⁺ condition makes pH_i acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pH_i. These results suggest that the reduced Ca²⁺ requirement of transformed cells for growth is related to the mechanism of pH_i regulation rather than Ca²⁺ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor modulates extracellular Ca2+ requirement for multiplication of normal human skin fibroblasts.

Epidermal growth factor (EGF) at 10 ng/ml reduces by over 50-fold the extracellular Ca²⁺ required for multiplication of normal human skin fibroblasts. Therefore, a Ca²⁺-related process may play a central role in the mechanism by which EGF exerts its effect on cell multiplication.     

Calmodulin and Ca2+ in normal and transformed cells

Numerous lines of evidence implicate calcium and calmodulin (CaM) as regulators of cell growth and functional differentiation. In light of this evidence, several studies of the possible involvement of the CaM system in cellular transformation by RNA and DNA tumor viruses have been carried out. This paper summarizes the evidence linking calcium and CaM to the regulation of cell growth and critically examines the evidence that increases in CaM levels occur in transformed versus normal cells. A nontraumatic method for synchronizing both normal and transformed chick fibroblasts is presented. This method is utilized in a comparison of CaM level throughout the cell cycle of Rous sarcoma virus transformed and normal chick embryo fibroblasts. These studies best support the hypothesis that the observed differences in CaM levels between transformed and normal cultures under optimal growth conditions may largely reflect differences in the proportion of cells in a dividing versus a nondividing state.     

Intracellular free Ca2+ changes during physiological polyspermy in amphibian eggs.

We have made the first measurements of intracellular free calcium activity ([Ca2+]i) in urodele eggs during physiological polyspermic fertilization. Jellied eggs of the urodele amphibian Pleurodeles waltlii were impaled with intracellular Ca(2+)-selective microelectrodes and inseminated under various conditions of sperm:egg ratio to obtain various degrees of polyspermy. In 17 out of 45 cases the egg [Ca2+]i level (0.41 microM) showed no variation following fertilization. In 28 other cases, however, the egg displayed a slow increase in [Ca2+]i of 0.15 microM, starting around 15 minutes after fertilization and reaching a plateau level around 10 minutes later. The amplitude of the fertilization-associated increase in [Ca2+]i was found to be independent of the number of sperm interacting with the egg surface. Measurements with two Ca(2+)-microelectrodes impaled in single eggs showed that the increase in [Ca2+]i did not simultaneously occur at distinct places within the egg cortex and, in some cases, could be detected by only one of the two microelectrodes. This latter observation, as well as the absence of [Ca2+]i change at fertilization in some experiments, strongly suggested that each sperm interacting with the egg might, at various places, trigger a localized, non-propagating change in [Ca2+]i. Experiments in which eggs were locally inseminated, using a micropipette directed towards the site of impalement of one of the two Ca(2+)-microelectrodes, clearly established that [Ca2+]i changes, although incapable of propagating over the entire egg cortex, might nevertheless travel very slowly over short distances, their amplitude vanishing rapidly as they propagate from around the sites of sperm entry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular Ca2+ concentration and latency of light-induced Ca2+ changes in photoreceptors of the honeybee drone

We have measured Ca_i at rest and upon light stimulation in the photoreceptors of the honeybee drone microfluorometrically with the fluorescent Ca²⁺ indicator dyes fura-2, fluo-3 and Ca-green 5N.In darkness, Ca_i was 90 nM after 5 min of dark adaptation. A saturating light step caused Ca_i to rise in the bulk cytoplasm to 750 nM within 1 s. Our measurements with the low affinity dye Ca-green 5N showed that bright 1-s light flashes cause a rapid increase in Ca_i which was graded with stimulus intensity. Ca-green 5N fluorescence reached a peak in about 200 ms, and then decayed to a slightly lower sustained plateau. The fluorescence signal peaked, when the receptor potential was repolarizing from its peak to the plateau. This observation is in agreement with the proposal that the peak-to-plateau transition of the receptor potential is caused by the rise in Ca_i From our Fluo-3 measurements it appears that the latency of the Ca²⁺ increase is by 3–4 ms longer than the latency of the receptor potential elicited by bright 100-ms light flashes. This result provides no support for the proposal that Ca²⁺ mediates the opening of those membrane channels responsible for the upstroke of the receptor potential.Abbreviations ER endoplasmic reticulum - IP₃ Inositol 1,4,5-trisphosphate - SMC submicrovillar cisternae     

Methionine biosynthesis in normal and transformed fibroblasts.

SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N⁵-methyltetrahydrofolate-homocysteine transmethylase and N^5–10-methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

Energy-dependent accumulation of Ca2+ by human embryonic lung fibroblasts.

Human embryonic fibroblasts accumulate Ca2+ in the presence of extracellular ATP and Mg2+, the uptake being maximal at 3 mM ATP. Iodoacetic acid, oligomycin and temperatures of 2 degrees C all inhibit the ATP-potentiated uptake suggesting that an active process may be involved in the transport of Ca2+ into these cells under certain conditions.     

Epidermal growth factor modulates extracellular Ca requirement for multiplication of normal human skin fibroblasts

Epidermal growth factor (EGF) at 10 ng/ml reduces by over 50-fold the extracellular Ca²⁺ required for multiplication of normal human skin fibroblasts. Therefore, a Ca²⁺-related process may play a central role in the mechanism by which EGF exerts its effect on cell multiplication.     

Growth-coupled changes in glucosaminoglycans (heparan sulfate and hyaluronic acid) in normal and transformed human fibroblasts

Changes in glycosaminoglycans (GAGs) were investigated in relation to cell density, growth and transformation of human fibroblasts. Relative amounts (percentages of the total GAGs) of heparan sulfate (HS) increased and those of hyaluronic acid (HA) decreased in growth-reduced (serum-starved, exogenous HS-treated and dense) cultures of normal (WI-38) cells. In contrast, transformed (WI-38 CT-1) cells exerted such GAG changes only in serum-starved cultures, but not in HS-treated or dense cultures. These results indicate that the changes in glucosaminoglycans (G1cAGs) (HS and HA) is coupled exclusively with cell growth.     

Involvement of intermediary metabolites in the pathway of extracellular Ca2+-induced mitogen-activated protein kinase activation in human fibroblasts.

Human fibroblasts in culture will grow in serum-free medium containing serum replacement factors, but without protein growth factors, as long as the Ca2+ level is 1.0-2.0 mM. When the Ca2+ is reduced to 0.1 mM, the cells stop cycling, but they can be reinduced to cycle by raising the Ca2+ level to 1.0 mM Ca2+ or to higher concentrations that result in activation of mitogen-activated protein kinase (MAPK). We now report that exposure of human fibroblasts to extracellular Ca2+ increased the level of inositol (1,4,5)-trisphosphate in the cytoplasm and caused a transient rise in the concentration of intracellular free Ca2+. Ca2+-induced MAPK activation was partly abolished by treatment of the cells with pertussis toxin. It was also decreased by treatment of cells with thapsigargin, which depletes intracellular Ca2+ stores; with phorbol 12-myristyl 13-acetate (PMA), which down-regulates protein kinase C (PKC); with the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide HCl (W-7), and calmidazolium (24571); as well as with lanthanum, a Ca2+ channel inhibitor. Ca2+ stimulation did not result in phosphorylation of the c-raf-1 protein. Our results suggest that extracellular Ca2+ stimulates MAPK activation through a pathway(s) involving a pertussis toxin-sensitive G protein, phospholipase C, intracellular free Ca2+, calmodulin, and PKC.     

Store-operated Ca2+-channels are sensitive to changes in extracellular pH

The sensitivity of store-operated Ca(2+)-entry to changes in the extra- and intracellular pH (pH(o) and pH(i), respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca(2+)-stores were depleted either with 1 mM carbachol (CCH) or with 2 microM thapsigargin (TG). Extracellular acidification suppressed both the CCH- and TG-mediated Ca(2+)-entry while external alkalinization augmented both the CCH- and the TG-induced Ca(2+)-influx. Mn(2+)-quenching experiments revealed that the rates of Ca(2+)-entry at the thapsigargin- or carbachol-induced plateau were both accelerated at pH(o) 8.2 and slowed down at pH(o) 6.8 with respect to the control at pH(o) 7.4. Alteration of pH(o) between 6.8 and 8.2 did not have any significant prompt effect on pH(i) and changes in pH(i) left the CCH-induced Ca(2+)-entry unaffected. These findings demonstrate that physiologically relevant changes in pH(o) affect the store-operated Ca(2+)-entry in SH-SY5Y cells and suggest that endogenous pH(o) shifts may regulate cell activity in situ via modulating the store-operated Ca(2+)-entry.     

Manganese superoxide dismutase in normal and transformed human embryonic lung fibroblasts

The manganese superoxide dismutase (MnSOD) activity of W138 human embryonic lung fibroblasts and SV40-transformed WI38 cells was measured. Under various growth conditions, the transformed cells always had lower MnSOD activity than their normal cell counterparts. The activity of MnSOD changes greatly with the growth conditions in the WI38 cells, while the MnSOD activity in the tumor cells remained more constant. The amount of immunoreactive MnSOD was measured by Western blotting. In all cases studied, the amount of immunoreactive MnSOD was lower in the transformed cells than in the normal cells.     

The effects of Ca2+ and Sr2+ on Ca2+-sensitive biochemical changes in human erythrocytes and their membranes.

1. The Ca2+-dependency of K+ efflux, microvesiculation and breakdown of polyphosphoinositides and of ankyrin have been measured in intact human erythrocytes exposed to ionophore A23187 and HEDTA [N'-(2-hydroxyethyl)ethylenediamine NNN'-triacetate]-Ca2+ buffers. Half-maximal responses were observed at pCa values of 6.4, 4.1, 5.0 and 4.8 respectively. 2. The Ca2+ dependencies of K+ efflux and breakdown of polyphosphoinositides and ankyrin measured in erythrocyte ghosts without addition of ionophore showed almost identical values with those seen in whole cells treated with ionophore. 3. We conclude that ionophore A23187 is able to cause rapid equilibration of extracellular and intracellular [Ca2+] in intact cells and that in the presence of a suitable Ca2+ buffer, ionophore A23187 can be used to precisely fix the intracellular concentration of Ca2+ in erythrocytes. 4. The relatively high concentration of Ca2+ required to produce microvesiculation in intact cells may indicate that microvesiculation could be at least partly dependent on a direct interaction of Ca2+ with phospholipid. 5. Results obtained with Sr2+ paralleled those with Ca2+, although higher Sr2+ concentrations were required to achieve the same effects as Ca2+. Mg2+ produced none of the changes seen with Ca2+ or Sr2+.     

Intracellular and extracellular changes of [Ca2+] in hypoxia and ischemia in rat brain in vivo

Changes in intra- and extracellular free calcium concentration were evaluated with ion-selective microelectrodes during periods of anoxia and ischemia in three different regions of intact rat brain. Recordings stable for at least 2 min and in most cases for 4-6 min were chosen for analysis. Under normoxic conditions neuronal [Ca2+]i varied between less than 10(-8) and 10(-7) M from cell to cell but no systematic regional differences were observed. Elimination of O2 or interruption in blood flow caused, within 30-60 s, slight intracellular alkalinization followed by a small rise in [Ca2+]i, a mild degree of hyperpolarization, and disappearance of electrical activity in the cortex, in that order. It is postulated that a decline in cellular energy levels, as manifested by H+ uptake associated with creatine phosphate hydrolysis, leads to an increase in [Ca2+]i, which activates Ca2(+)-dependent K+ channels and consequently enhances gK. 2-4 min later there was a sudden, large rise in [K+]e, a fall in [Ca2+]e and a rapid elevation of [Ca2+]i. The magnitude of the latter was greatest in a high proportion of hippocampal neurons in area CA1 and some cortical cells, while it was smallest and relatively delayed in thalamic neurons. In the hippocampus area CA1 increases in [Ca2+]i to as much as 6-8 x 10(-4) were observed; some of these could be reversed when O2 or blood flow were restored to normal. Pretreatment of animals with ketamine and MK-801, antagonists of excitatory amino acid transmitters, markedly slowed and decreased the rises in [Ca2+]i. The effects of the two agents were most pronounced in the hippocampus. It is concluded that the receptor-operated channels are largely responsible for Ca2+ entry into certain cells during hypoxia/ischemia. This pathway may be of primary importance in parts of the hippocampus and cortex, regions of the brain that are particularly vulnerable to O2 deprivation and which receive high glutamatergic input and have an abundance of excitatory amino acid receptors.     

Vimentin-derived proteins : Differences between normal human fibroblasts and transformed human cells

Two-dimensional gel electrophoresis revealed a quantitative difference in a series of polypeptides ranging in MW from 45 000 to 51 000 and of lower isoelectric pH than vimentin, when comparing normal human fibroblasts with a virally transformed subline and with HeLa cells. Re-extraction of purified [³⁵S]vimentin with cold whole cell homogenates and peptide mapping showed that these polypeptides are derivatives of vimentin. They may be natural components of a normal fibroblast's architecture or they may arise from a pool of vimentin that is not structured within intermediate filaments at the time of extraction. Furthermore, we show that vimentin from the two transformed cell types is more resistant to proteolysis by whole cell homogenates than vimentin from normal fibroblasts. Structural alteration of vimentin may play an important role in the expression of transformation.