The mechanism of action of FK 506.
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Amino Acid Isomerases↗
Biomedical subjects
J E Kay
Publications and source records attributed to J E Kay.
Control of protein synthesis by extracellular Na+ in cultured fibroblasts.
In chick embryo fibroblasts (CEFs), a partial substitution of extracellular Na+ with other cations or carbohydrates decreased the intracellular Na+ content without altering the K+ level. Concomitantly, a significant decrease in the serum-dependent rate of protein synthesis occurred. This phenomenon appeared to be quickly reversible upon reconstitution of the correct extracellular Na+ concentration in the culture medium. The presence of a transcriptional inhibitor such as actinomycin D during the treatment did not inhibit the reversibility of the phenomenon. The presence in the culture medium of K+ in such excess as to dissipate the membrane potential did not alter the observed relationship between the protein synthesis rate and the internal Na+ content. Analysis of the amino acid pool indicated that the observed inhibition of the rate of protein synthesis in CEFs incubated in low Na+ medium was not caused by an unbalanced availability of intracellular amino acids. In addition, intracellular pH, as estimated by the measurement of the equilibrium distribution of benzoic acid, did not show any significant alteration in cells incubated in the presence of bicarbonate buffer and in low extracellular Na+. Moreover, the relationship between the rate of protein synthesis and the internal Na+ content was still observed in CEFs cultured in bicarbonate-containing media, but at lower or higher than physiological pH. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of the proteins synthesized by CEFs cultured at a reduced extracellular Na+ concentration showed that specific alterations of gene expression occurred.
Amino Acids↗
The mechanism of action of the immunosuppressive drug FK-506.
The novel immunosuppressive drug FK-506 inhibits the induction of lymphocyte proliferation in vitro by mitogenic combinations of phorbol esters and calcium ionophores. Early events inducible by phorbol esters alone are unaffected, while changes induced by calcium ionophores alone are completely suppressed by as little as 0.1 nM FK-506. The event in lymphocyte activation inhibited by FK-506 is completed early in the response. Its completion requires the provision of a Ca2+ signal and concurrent activation of protein kinase C and is accelerated as a function of the strength of protein kinase C activation.
Animals↗
T lymphocyte activation through the C28 pathway is insensitive to inhibition by the immunosuppressive drug FK-506.
Nanomolar concentrations of the novel immunosuppressive drug FK-506 inhibit the proliferation of human T lymphocytes in vitro induced by mitogenic lectins or by monoclonal antibodies directed against the CD3 or CD2 surface antigens. However, the alternative pathway of T lymphocyte proliferation induced by monoclonal antibodies specific for CD28 together with phorbol esters is unaffected by FK-506.
Anti-Bacterial Agents↗
Inhibition of T-lymphocyte activation by the immunosuppressive drug FK-506.
Nanamolar concentrations of the immunosuppressive drug FK-506 inhibit the induction of T-lymphocyte proliferation by the lectins concanavalin A (Con A) and phytohaemagglutinin (PHA). Activation by Con A is more sensitive to inhibition than the response to PHA. FK-506 inhibits an early Ca2+-dependent step in the activation process, and its effects are not reversible by the addition of recombinant interleukin-2 (IL-2) or lymphokine-rich culture supernatant. While the effects of suboptimal concentrations of FK-506 and cyclosporin A (CsA) are additive, FK-506 does not enhance the effects of optimal concentrations of CsA. Both drugs also have similar effects on the expression of specific mRNA in Con A-activated lymphocytes. A brief preincubation of unstimulated cells with FK-506 irreversibly inhibits their subsequent responsiveness to Con A. The mechanism of action of FK-506 thus resembles that of CsA, except that it is effective at two to three orders of magnitude lower concentrations and its effects are much less readily reversible.
Animals↗
Inhibition of murine B-lymphocyte proliferation by the novel immunosuppressive drug FK-506.
The novel immunosuppressive drug FK-506 inhibits both cell-mediated and humoral immunity in vivo and suppresses T-lymphocyte activation at nanamolar concentrations in vitro. We report here that FK-506 also inhibits the in vitro proliferation of murine B lymphocytes induced by goat antimouse IgM (GaMIgM), although it does not affect the induction of proliferation by bacterial lipopolysaccharide (LPS). The action of FK-506 on B-lymphocyte proliferation thus resembles that of cyclosporin A, further strengthening the correlation between the action of the two immunosuppressive drugs previously reported for T-lymphocyte activation.
Animals↗
Adaptive response of cultured fibroblasts to hyperosmolarity.
Raising to 0.4 osM the osmolarity of the medium in which chick embryo fibroblasts are incubated quickly increases the internal Na+ concentration, inhibits protein synthesis and also stimulates amino acid transport. On extending the incubation time, cells appear to adapt to the altered environment, as the Na+ content declines toward control values within few hours. Protein synthesis resumes its normal rate within 12-14 h of treatment. Experimental alteration of the monovalent cation content by substituting extracellular Na+ with other osmolites or by using ouabain or the ionophore monensin reveals an impairment of protein synthesis. Analysis by SDS-PAGE reveals an alteration of the polypeptide pattern expressed by hyperosmolarity-exposed cells, resulting in an enhanced synthesis of the 87, 75 and 53 kD proteins and inhibition of a 125 kD band. The previously increased amino acid transport activity also reverts to its normal level, but only after 40-50 h of incubation. The growth rate of these cells does not appear to be significantly affected during the first 3 days of the hyperosmolar treatment. Results presented in this publication identify the alteration of the protein synthesis rate, the change in the intracellular cation content and the increase in amino acid transport activity as plausible parameters of adaptive response, and suggest that the modulation of gene expression observed in cells exposed continuously to hyperosmolarity may be a consequence of the alteration of the intracellular monovalent cation concentration.
Adaptation, Physiological↗
The role of the transferrin receptor in lymphocyte activation.
Blockade of the transferrin receptors whose expression is induced in lymphocytes incubated with the mitogenic lectin phytohaemagglutinin (PHA) does not affect the initial stimulation of protein synthesis but does strongly and progressively inhibit the subsequent induction of DNA synthesis. When the effects of transferrin receptor blockade on the induction of the enzymes uridine kinase (whose induction begins early in G1 phase of the cell cycle) and thymidine kinase (whose induction is closely associated with DNA synthesis) were examined, both enzymes were found to be induced normally. This indicates that the function of the transferrin receptor is directly to provide a component essential for DNA synthesis itself (probably iron) rather than to act as the receptor for a general signal required to initiate entry into S-phase.
Antibodies, Monoclonal↗
Lymphocyte activation by OKT3: cyclosporine sensitivity and synergism with phorbol ester.
Lymphocyte activation by the mitogenic monoclonal antibody OKT3 is less effective than activation by mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A). Activation by OKT3 is also very sensitive to inhibition by cyclosporine (CSA), which selectively inhibits Ca2+-activated steps in the activation process. In addition, the magnitude of the OKT3 response can be raised to that seen with mitogenic lectins by coincubation with phorbol esters (which activate protein kinase C). These observations suggest that OKT3 may deliver efficiently the Ca2+ signal involved in the initiation of lymphocyte activation, and that the comparatively weak overall response is due to a failure to generate a second signal, probably the activation of protein kinase C, as efficiently as the mitogenic lectins.
Antibodies, Monoclonal↗
The effects of prostaglandin E and I analogues on lymphocyte stimulation.
The prostacyclin analogue carbacyclin and the prostaglandin E analogues hydantoin no. 245C77 and no. 454C77 were potent inhibitors of mitogen-stimulated DNA and protein synthesis by cultured pig peripheral blood lymphocytes with ID50 concentrations between 10(-5) and 10(-8)M. These effects were comparable to those of PGE2. Strong inhibition was observed only with carbacyclin when human lymphocyte cultures were used. These results show that stable prostaglandin analogues may exhibit at least some of the activities of the naturally occurring PGE2 in this system, and support the concept that prostacyclins may also play a role in regulating immunological responses.
Dinoprostone↗
Rapid loss of sensitivity of mitogen-induced lymphocyte activation to inhibition by cyclosporin A.
The ability of the immunosuppressive drug cyclosporin A (CS-A) to inhibit the activation of lymphocytes by phytohaemagglutinin (PHA) and concanavalin A (Con A) is progressively lost over the 8-hr period following mitogen addition. This process is dependent on the presence of Ca2+ in the culture medium and is complete at a time when activation still requires the continued presence of the mitogen. While inhibition by CS-A is reduced to some extent by lymphokines produced by mitogen-activated cultures, the initial loss of sensitivity to CS-A is too complete and too rapid to be accounted for in this way. We conclude that CS-A inhibits an early Ca2+-dependent step in mitogen-induced activation that is not in itself sufficient to commit the cells to initiate proliferation, but is required for later steps in the activation process, including lymphokine production.
Animals↗
Early events during the activation of human lymphocytes by the mitogenic monoclonal antibody OKT3.
The effects of the mitogenic monoclonal antibody OKT3 on the metabolic changes preceding DNA synthesis during the activation of human peripheral blood mononuclear cells were compared with those induced by PHA. The aspects studied included uridine transport, the incorporation of inositol into phospholipids, Na+-dependent amino acid uptake, and protein synthesis. All four parameters were increased in response to the ligation of the T lymphocyte receptor recognized by OKT3. These changes were apparent as early as the corresponding changes induced by PHA. However, the increases in uridine uptake and inositol incorporation were disproportionately reduced when compared to those caused by PHA, and no evidence of high-dose inhibition was seen in cells activated by OKT3. This suggests that at least some lectin-induced changes in metabolism are mediated through additional mechanisms, probably involving distinct receptors.
Alanine↗
The effects of indomethacin on T lymphocyte stimulation.
Immunomodulatory effects of indomethacin on mitogen-induced [3H]thymidine incorporation in cultured pig peripheral blood lymphocytes were demonstrated. In monocyte-containing lymphocyte cultures, indomethacin enhanced lymphocyte stimulation, whereas in monocyte-depleted cultures indomethacin inhibited the response. Indomethacin effects were most marked at suboptimal to optimal concentrations of mitogen and decreased at superoptimal concentrations in both types of culture. The effects of prostaglandin E2 at superoptimal mitogen concentrations were found paradoxically to be stimulatory. The results indicate that arachidonic acid metabolites may have both positive and negative effects on lymphocyte activation.
Animals↗
Activation of T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate is resistant to inhibition by cyclosporin A.
Cultured T lymphocytes from pig blood can be activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Activation is additive with that induced by the mitogenic lectin phytohaemagglutinin (PHA). Activation by TPA differs from that induced by PHA or other mitogenic lectins in that it is not inhibited even by high concentrations of the immunosuppressive drug cyclosporin A (CS-A). Neither co-culture of lymphocytes with PHA and TPA nor addition of culture supernatants from TPA-stimulated cultures affected the sensitivity to CS-A of the response to PHA.
Animals↗
Effect of cyclosporin A on lymphocyte activation by the calcium ionophore A23187.
The activation of pig lymphocytes by the divalent cation ionophore A23187 is more sensitive to inhibition by cyclosporin A than is activation by plant lectins such as concanavalin A. Complete inhibition of A23187-induced activation was seen at a cyclosporin A concentration of 0.3 micrograms/ml. The very early stimulation of nucleoside uptake induced by A23187 was not affected by cyclosporin A, but other early metabolic changes occurring during the first few hours after activation were inhibited.
Alanine↗
Effects of cyclosporin A on the metabolism of unstimulated and mitogen-activated lymphocytes.
The immunosuppressive drug cyclosporin A (CS-A) reduces the magnitude of T-lymphocyte activation by all mitogenic lectins tested. However, in all cases a proportion of the activation observed is resistant even to very high concentrations of the drug. This proportion depends on the mitogen used, the responses to concanavalin A (Con A) and soybean agglutinin (SBA) being much more strongly inhibited than the responses to phytohaemagglutinin (PHA) or pokeweed mitogen (PWM). The differential effects of CS-A on lymphocyte activation by these mitogens could not be accounted for by the magnitude of the mitogenic response, the mitogen concentration used or the dependence of the responses on the presence of accessory cells, and they were maintained when several different procedures were used to assess the degree of activation. CS-A effectively inhibited inhibited lymphocyte activation. CS-A effectively inhibited lymphocyte activation only when added prior to, or very shortly after, the mitogen. Its ability to inhibit the response to PHA was lost more rapidly than that of Con A. The rate of protein synthesis by unstimulated lymphocytes was also affected by CS-A over the concentration range required to inhibit activation by mitogens. Although this effect was smaller than the inhibition of mitogen activation, it was highly significant and reproducible, and could not be accounted for by inhibition of spontaneous activation occurring in the unstimulated cultures.
Animals↗
Initiation reactions in the mRNA-dependent reticulocyte lysate.
Reticulocyte lysates depleted of mRNA by digestion with micrococcal nuclease still show an unexpectedly high rate of formation of 80 S initiation complexes. Formation of these complexes is sensitive to all inhibitors of the normal protein synthesis initiation process tested. Such lysates contain high concentrations of mRNA fragments which can be utilized for initiation, with which exogenous mRNA must compete. As a consequence of this competition, mRNAs that are weak initiators may be translated poorly by this system even at low exogenous mRNA concentrations.
Animals↗
Translational regulation of protein synthesis in eukaryotes.
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Animals↗