A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells.

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cell whole sonicate or cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.

Antithymocyte gamma globulin, low-dosage cyclosporine, and tapering steroids as an immunosuppressive regimen to avoid early kidney failure in heart transplantation.

Cyclosporine is a powerful immunosuppressive agent that unfortunately has significant renal toxicity. Two risk factors associated with a high incidence of kidney failure in patients receiving cyclosporine have been described in the literature. In an effort to decrease the possibility of renal toxicity with the use of cyclosporine, we use low-dosage cyclosporine, antithymocyte gamma globulin, and tapering dosages of steroids as an immunosuppressive regimen. Twenty-one patients had orthotopic heart transplants from January 1985 to January 1986. Sixteen of 21 patients or 70% had at least one high risk factor for kidney failure. There were no episodes of acute kidney failure, and the blood urea nitrogen and creatinine levels that were recorded over an average of 8.5 months per patient did not increase significantly from preoperative values. Seventeen of 21 or 81% of the patients are alive and functioning fully. The incidence of rejection per patient was 0.9, and there were no biopsy-proven severe rejections. One patient died at 5 months; the autopsy showed generalized moderate rejection. There were 0.24 episodes of infection per patient, with one patient who died from Pneumocystis pneumonia. With this immunosuppression protocol, early postoperative kidney dysfunction was avoided. The incidences of rejection and infection were within acceptable range, and the quality of life in the 17 survivors is excellent.

Further characterization of the effect of bacterial lipopolysaccharide preparations on cyclic GMP levels: the importance of macromolecular synthesis.

Bacterial lipopolysaccharides (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver cells without affecting the concentration of cAMP. This effect is produced by very small (1 ng) amounts of LPS and is both dose and time dependent. The time dependence is characterized by an initial lag period of 60-120 min followed by a rapid, persistent increase in cGMP levels. Since this time course suggests that synthesis of an intermediate might play an important role in the cGMP elevation, a series of experiments was done to evaluate the effect of LPS on DNA, RNA, and protein (macromolecular) synthesis. LPS did not measurably effect total macromolecular synthesis. However, inhibitors of RNA and protein synthesis markedly reduced cGMP levels in LPS-treated cells, whereas inhibition of DNA synthesis did not. Addition of sodium nitroprusside to control and inhibitor-treated cultures produced large equivalent increases of cGMP levels in both cases, indicating that the cells present were fully capable of responding to a stimulus of guanylate cyclase. Taken together, this data suggests that expression of the LPS-cGMP response in fetal liver cells is dependent on synthesis of an intermediary protein(s) during the lag phase.

Further characterization of the effect of bacterial lipopolysaccharide preparations in cyclic 3',5'-GMP levels: the importance of serum.

Bacterial lipopolysaccharides (LPS) greatly increase cGMP levels in rat fetal liver cells without affecting the concentration of cAMP. This elevation is due to the Lipid A moiety of the LPS molecule, is time and dose dependent, and is markedly potentiated by small amounts of serum. Because of the magnitude of the serum potentiation, a series of experiments was undertaken to further characterize this effect. Although serum was not absolutely necessary for a cGMP response, small amounts increased the cGMP potency of LPS more than 100-fold. Under these same conditions, serum did not greatly affect the ability of nitroprusside to raise cGMP levels or of epinephrine to raise cAMP levels. The effect of serum on the cGMP response was dose dependent and was produced with as little as 1 microliter (0.1% v/v) per culture. Absorption with limulus lysate, heating to 56 degrees C for 30 min, or extensive dialysis did not significantly alter the cGMP enhancing effect of serum. However, albumin and IgG were both inactive at all concentrations tested. Thus, serum produces a large dose dependent enhancement of the LPS induced cGMP response that appears to be selective, i.e. it is not a non-specific protein effect and there is no similar potentiation of nitroprusside stimulated cGMP levels. Furthermore, the cGMP effect of serum is probably not related to substances that opsonize bacteria and is due to a component or components of serum large enough not to be dialyzable.