Interaction of far- and near-ultraviolet radiation. The occurrence of photo-augmentation and photo-recovery in cultured mammalian cells

Guided by the phenomena of photo-augmentation and photo-recovery, which have been described with respect to the induction of erythema in human skin, experiments were undertaken with cultured mammalian cells to study whether irradiation with far- and near-ultraviolet radiation results in an interaction at the cellular level with respect to cell survival and induction of mutations. Evidence was found for both photo-augmentation and photo-recovery. Photo-augmentation (more than an additive effect) was observed for cell survival when the long-wave ultraviolet irradiation (UVA) preceded the short-wave ultraviolet irradiation (UVB). Photo-recovery (less than an additive effect) was observed for cell survival if the UVA was given after or simultaneously with the UVB. The latter effect, however, was strongly influenced by dose: doses of UVA higher than 20 000 J/m2 no longer lead to photo-recovery in cell survival. For mutation induction, reduction in mutant frequency appears indicated for both combinations of UVA and UVB and for high and low doses of UVA.     

Selectivity in metabolic cooperation between cultured mammalian cells

Selective communication between cultured mammalian cells was detected as selectivity in metabolic cooperation. Whilst the majority of the cell types examined (human skin fibroblast, PC13, G3, Don, PyY) showed metabolic cooperation at almost all (>95%) of their homotypic cell-to-cell contacts, they did not necessarily show cooperation at such a high proportion of their heterotypic contacts. Less than 10% of G3/human fibroblast contacts, and usually less than 30% of G3/PC13 contacts were observed to be positive for metabolic cooperation. L cells differed from these other cell types in that they formed permeable junctions at a greater proportion of their heterotypic cell-to-cell contacts (contacts between L and PyY cells) than their homotypic contacts. We question why it was that the contacts between any two poorly-compatible cell types were positive for metabolic cooperation in only a small proportion of cases. We could find no indication that this phenomenon was attributable to heterogeneity within the cell stocks. Time course studies upon PC13 and G3 cells showed that the proportion of cooperation-positive contacts was not constant but that it continued to increase over many hours of co-culture. In comparing the homotypic and heterotypic interactions of these cell types, selectivity manifested as a difference in the rate of appearance of permeable junctions. We discuss possible explanations for these findings.     

The effects of dihydropyridines on neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of (3H) norepinephrine and (3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of (3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.     

Relative effects of cooling and warming rates on mammalian cells during the freeze-thaw cycle

The relative roles of cooling and warming rates on cell survival during a freeze-thaw cycle were investigated. Basically the faster the warming rate, the better the cells survive. One of the factors influencing this is the extended phase transition period at the slower thawing rates. The warming rate had a significant effect on cell damage and recovery, but this was not as great as comparative changes in the cooling rate were. This investigation also showed that under certain freeze-thaw conditions there was a lack of correlation between the two methods used for quantifying cell recovery (RI) and cell damage (PCR) as measured by radiochromate release. The analysis of the relationship between RI and PCR showed that PCR could be used to measure both lethal and nonlethal damage and enabled a clearer interpretation of cellular damage during cooling and thawing to be made.     

Effect of sulfhydryl reagents on nucleoside transport in cultured mammalian cells

Incubation of Novikoff rat hepatoma cells; mouse L929, P388 and L1210 cells; and Chinese hamster ovary cells with sulfhydryl reagents, such as p-hydroxymercuribenzoate or p-hydroxymercuribenzenesulfonate, reduced the zero-trans influx of uridine in a concentration-dependent manner. The sensitivity of uridine transport to inhibition varied somewhat for the cell lines, Chinese hamster ovary cells being the most sensitive. Maximum inhibition by p-hydroxymercuribenzoate occurred in 10–20 min of incubation at 37 °C, and was associated with a decrease in maximum transport velocity without significant change in substrate affinity of the carrier. The development of inhibition of uridine influx correlated with binding of [¹⁴C]p-hydroxymercuribenzoate to the cells. Inhibition of transport also roughly correlated with a decreased binding of 6-nitrobenzylthioinosine to high-affinity binding sites on the cells (presumably representing the nucleoside transporter) without affecting binding affinity. Treatment of cells with p-hydroxymercuribenzenesulfonate reduced uridine influx and efflux to a similar extent. Inhibition of uridine transport and binding of [¹⁴C]p-hydroxymercuribenzoate were readily reversed by incubation of the cells with dithiothreitol. The results indicate that sulfhydryl groups are essential for the functioning of the nucleoside transporter, perhaps for the binding of substrate. Blockage of the sulfhydryl groups results in a reversible inactivation of the carrier. Treatment of the cells with the sulfhydryl reagents also caused a concentration-dependent increase in cell volume, which was readily reversed by incubation of the cells with dithiothreitol but seemed unrelated to the inhibition of nucleoside transport.     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Persistence of exogenous, inserted ganglioside GM1 on the cell surface of cultured cells

Ganglioside GM₁, which can insert spontaneously into the membrane of intact cells, has been measured after insertion into transformed fibroblasts by cholera toxin (choleragen) binding, for which ganglioside GM₁ is the natural receptor. Choleragen binding is not altered in starved, quiescent cells over a four-day period. Dividing cells show decreased binding in proportion to cell division. Thus, neither dividing nor quiescent cells appear to metabolize or otherwise degrade this membrane component.     

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

2'-deoxyadenosine-3'-monophosphate: A naturally occurring inhibitor of adenylate cyclase in amphibian and mammalian cells

A naturally occurring inhibitor of adenylate cyclase has been found in various rat tissues and toad erythrocytes. The compound containing this inhibitory activity has been isolated from several cell types. Physical, chemical, and enzymatic analyses define this inhibitor as 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP). Measurements of the effect of various nucleoside monophosphates on adenylate cyclase activity reveal that the inhibitory activity is specific for 2'-deoxy-3'-AMP. In addition, the metabolism of this inhibitor may be relatively specific since only aminophylline and cAMP appear to inhibit the breakdown of 2'-dexy-3'-AMP by liver microsomes. The presence of this inhibitor suggests the possibility that deoxynucleotides may play an important role in the action of hormones and the regulation of intracellular metabolism.     

The effects of ketamine, phencyclidine and lidocaine on catecholamine secretion from cultured bovine adrenal chromaffin cells

The ability of ketamine, phencyclidine and analogues to alter catecholamine secretion from cultured bovine adrenal chromaffin cells was investigated. Both ketamine and phencyclidine specifically inhibited nicotinic agonist-induced secretion at concentrations which did not alter secretion induced by elevated K+ depolarization. The inhibition of nicotinic agonist-induced secretion was not overcome by increasing concentrations of nicotinic agonist. The effects of stereoisomer pairs of phencyclidine-like drugs - dexoxadrol, levoxadrol and (+)PCMP, (-)PCMP - did not reveal stereospecificity for the inhibition, in contrast to the stereospecific behavioral effects of the drugs. The local anesthetic lidocaine (0.3 mM) also noncompetitively inhibited nicotinic agonist-induced secretion without inhibiting elevated K+-induced secretion. The data indicate that ketamine and phencyclidine at clinically relevant concentrations specifically inhibit the adrenal chromaffin cell nicotinic receptor at a site similar to or identical with the site of action of local anesthetic. Although the nicotinic receptor inhibition is probably not related to the anesthetic and behavioral effects of ketamine and phencyclidine, it is likely that the centrally mediated increase in sympathetic nervous system activity which is characteristic of these drugs is moderated by the peripheral blocking effects on catecholamine secretion from the adrenal medulla.     

2-Pyrrolidinone and succinimide endogenously present in several mammalian species

2-Pyrrolidinone and succinimide were identified in blood plasma of man, rat, and mouse. Dog plasma contained only traces of 2-pyrrolidinone not exceeding significantly the detection limit of our GCMS-method. Succinimide but not 2-pyrrolidinone could also be found in the brains of rat and mouse. Evidence is presented for a metabolic pathway leading from 2-pyrrolidinone to succinimide, with 5-hydroxy-2-pyrrolidinone as an intermediate.     

Regulation of 1,25(OH)2 vitamin D3 receptor content in cultured LLC-PK1 kidney cells limits hormonal responsiveness

Receptor content in cultured kidney (LLC-PK1) cells was found to be modulated following the introduction of a culture medium change, declining to 40% of control values at 18 h. Scatchard analysis indicated that the reduced 1,25(OH)2-[3H]D3 nuclear binding we detected was due to decreased abundance of receptors (3811 vs 1619 sites/cell) with no change in the Kd (0.4-0.5 nM). Cells with reduced receptors exhibited diminished ability to respond to 1,25(OH)2D3 as measured by induction of 25(OH)vitamin D-24-hydroxylase activity. There was a close coupling between decreased receptor levels and diminished hormone responsiveness. The data suggest the absence of "spare" receptors and that receptor abundance is a limiting factor in cell responsiveness to 1,25(OH)2D3.     

Immunofluorescent and autoradiographic analysis of the relationship between the presence of histone H5 and DNA synthesis in developing chick erythroid cells

Simultaneous detection of histone H5 by indirect immunofluorescence and of [³H]thymidine incorporation by autoradiography on the same preparations of developing erythroid cells have been used to precisely define the extent of correlation between the loss of nuclear activity and the presence of histone H5. It was found that from day 3–12 of embryonic life there are two successive waves of double-labelled cells. At some stages, as many as 30% of the cells which incorporate [³H]thymidine also contain histone H5. Thus, the simple presence of H5 cannot be sufficient to cause nuclear inactivation. A kinetic analysis of the appearance and disappearance of [³H]thymidine-labelled cells, containing histone H5, and cells which are positive for both markers is presented. The result is consistent with the interpretation that the appearance of H5 in the first wave of double labelled cells occurs just before the erythroid cells become metabolically inactive. These observations modify the concept that histone H5 functions uniquely or solely as a template repressor.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

Dexamethasone induces increased catecholamine biosynthesis in cultured neuroblastoma

The effect of dexamethasone on morphological and biochemical parameters of catecholamine biosynthesis in cultured murine neuroblastoma cells was determined. Treatment for 7 days with 25 μM dexamethasone produced a more intense and uniform catecholamine fluorescence as determined by fluorescence histochemistry using the glyoxylic acid technique. Treatment with dexamethasone also produced a two-fold increase in dopamine content and a threefold increase in tyrosine hydroxylase activity compared with controls. These results suggest that this potent glucocorticoid induces differentiation in cultured murine neuroblastoma, as measured by both biochemical and morphological techniques.     

Evidence for selection in cultured diploid fibroblast strains

Single skin fibroblast cultures were established from 29 females heterozygous at the X-linked G-6-PD locus for Gd^B and an electrophoretic variant, Gd^A or Gd^A?. The cultures were transferred serially until they displayed single-enzyme phenotypes (only type A or type B enzyme) or until growth ceased. Single-enzyme phenotypes were observed in 19 instances. This occurred as early as the 4th, and as late as the 38th transfer. Ten cultures evolved A phenotypes and 9 developed B phenotypes. Possible explanations for the development of single-enzyme phenotypes include random cell-sampling during sub-culturing, selection or both. Selection occurring at least in part on the basis of X-linked loci other than G-6-PD seems most likely for those cultures which evolved rapidly to single-enzyme phenotypes, but sampling or selection or both could have played important roles in those cultures which maintained double-enzyme phenotypes for several passages and then rapidly developed single-enzyme phenotypes. In any event, the results indicate that it should not be assumed in investigations using cultivated diploid fibroblasts that the latter are generally representative of the tissue-as-a-whole from which the cultures originate.     

Cytotoxic effect of a T-strain mycoplasma (Ureaplasma urealyticum) on cultured normal human cells (WI-38)

Normal human embryonic lung fibroblasts (WI-38) were infected with Ureaplasma urealyticum, a urea hydrolysing mycoplasma. It was possible to observe reduced rates of multiplication of infected cells and reduced plating efficiency as well as the morphological changes usually associated with mycoplasma infection of animal cells in vitro. The cytotoxic effect on multiplication was sensitive to aureomycin but not penicillin. It was not related to depletion of amino acids or nucleic acid precursors from the cell culture medium but appeared to require that the host cells be growing. Ureaplasmas could not be recovered from cell culture medium after 4 days post infection and their characteristic urease activity could not be demonstrated either in cell culture medium or associated with the cells after the first cell subcultivation. [³H]TdR was incorporated into the nuclei of infected cells and the percent labelled nuclei was reduced compared with uninfected cells. Nuclear labelling indices of infected cells increased as the cells were subcultivated by trypsinization suggesting that the ureaplasmas were removed from the host cell surface by this treatment. In general, the effects of ureaplasmas on WI-38 cells do not appear to be as pronounced as effects of other mycoplasmas on animal cells in culture. It is clear, nonetheless, that the urea hydrolysing mycoplasmas can infect cells in culture and cause discernible effects on the growth and metabolism of these cells.