Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.

Anesthetics, Local↗

PubMed1979

Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells.

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.

Animals↗

PubMed1978

Retention of transformant specific type III collagen in dibutyryl cAMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant.

Explore the source record for details and available documents.

Bucladesine↗

PubMed1977

Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxide formation.

Several types of cultured fibroblasts, including chick embryo, human and mouse, were killed by the addition of sodium ascorbate at final concentrations of 0.05-0.25 mM to cultures at the time of inoculation or to attached cells. Ascorbate did not affect the attachment of cells to the substratum. The effect on chick embryo fibroblasts was visible by four hours and by six hours almost all cells had swelled and were becoming detached. By 24 hours detached cells had either lysed or become crenated in appearance. Other end-diol reducing agents and also glutathione and cysteine were effective while gulonolactone, a non-reducing analogue of ascorbate, was ineffective. Preincubation of medium containing ascorbate but no cells, conditions which result in degradation of the vitamin, led to loss of toxicity, indicating that a degradation product was not the lethal agent and that a component of the medium was not converted to a lethal substance. The lethal effect of both ascorbate and glutathione was prevented by the addition of catalase to the medium suggesting that H2O2 formed by intracellular reactions and then excreted into the medium was the cytotoxic agent. This conclusion was supported by the findings that 0.05 mM H2O2 added to chick embryo fibroblasts was lethal and that the effect of this compound on cellular morphology was almost identical to that of ascorbate.

Ascorbic Acid↗

PubMed1977

Specific changes in the collagen phenotype of BALB 3T3 cells as a result of transformation by sarcoma viruses or a chemical carcinogen.

Radioactive proline-labeled procollagen, accumulated during a 3-hr incubation of normal and transformed BALB 3T3 cultures, was treated with pepsin and the resulting collagen components were analyzed by carboxymethyl-cellulose chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the presence or absence of reducing agent. Collagen in the medium of three subclones of BALB 3T3 A-31 that exhibited contact-inhibition of growth at confluence, as well as in the medium of one that did not, consisted of alpha(1) and alpha(2) subunits in the ratio of 3:1, suggesting that 3T3 cells synthesize type I collagen, [alpha(1)(I)](2)alpha(2), and another type, which we have designated X, composed of alpha(1) chains, which may or may not be identical to alpha(1)(I). Culture medium from 3T3 transformed by Kirsten or Moloney sarcoma virus contained type I collagen and another type differing from I and X and designated as type Y. The latter appeared to be similar to type III collagen [alpha(1)(III)](3), since it contained intrahelical disulfide bonds. Analysis of intracellular collagen also demonstrated the presence of type III in Ki-3T3 and its absence from 3T3 cells. Collagen components from the medium of a simian virus 40 transformant were identical to those of the contact-inhibited clones, while the collagen from a 4-nitroquinoline-1-oxide-induced transformant was composed mainly of two components differing from alpha(1)(I), alpha(2), or alpha(1)(III). These results suggest that the type of collagen accumulated in transformed cell cultures may be specifically related to the transforming agent.

4-Nitroquinoline-1-oxide↗

PubMed1976

Submicrosomal localization of prolyl hydroxylase from chick embryo limb bone.

A fraction greatly enriched in microsomes was prepared from chick embryo limb bone tissue homogenates by differential centrifugation in a high density solution of Metrizamide. This fraction was used to determine the submicrosomal localization of prolyl hydroxylase. At a low concentration (0.05%) of the non-ionic detergents Triton X-100 and Brij-35, 90 to 93% of prolyl hydroxylase activity was released from microsomes. Concentrations of Triton X-100 greater than 0.1% were required to solubilize the intrinsic membrane enzyme NADH-ferricyanide reductase and to release membrane-bound ribosomes, while Brij-35 did not extensively solubilize membrane components even at concentrations up to 0.4%. In addition, prolyl hydroxylase activity which could subsequently be released from microsomes by Brij-35 was relatively resistant to trypsin proteolysis at concentrations which removed more than 50% of the ribosomes and approximately 40% of the protein from microsomes. These results suggest that 90 to 93% of prolyl hydroxylase activity in connective tissue is located within the cisternae of the endoplasmic reticulum. Gel filtration of prolyl hydroxylase released from microsomes or found in the soluble fraction of limb bone homogenates revealed two peaks of activity corresponding to molecular weights of 230,000 and 450,000 to 500,000. The latter is twice the value reported for purified chick embryo prolyl hydroxylase. A fraction of the total prolyl hydroxylase activity (generally 20 to 35%) in microsome preparations could be measured in the absence of detergent, although the microsomal membrane should be impermeable to the large unhydroxylated collagen chains used as substrate. On the basis of experimental data, it was concluded that detergent-independent activity was most likely due to damaged microsomal membranes and that this damage was sufficient to allow substrate and trypsin to enter the cisternae but not to allow prolyl hydroxylase to be released.

Animals↗

PubMed1976

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.

Ascorbic Acid↗

PubMed1975