Fig. 4. Effect of isoflurane on inducible nitric oxide synthase (iNOS) mRNA expression in immunostimulated murine J774 macrophages in the presence and absence of ionomycin shown by representative Northern blot results. (A ) Cells were either stimulated for 4 h with lipopolysaccharide (1 μg/ml; lanes 1, 3, 5, 7) or γ-interferon (γIFN; 200 U/ml; lanes 1, 2, 5, 6) or left unstimulated (controls, lanes 1 and 8) in the presence (lane 1–4) or absence (lanes 5–8) of 2 minimum alveolar concentration (MAC) isoflurane, as indicated. Similar results were obtained in three separate experiments. (B ) Antagonizing effect of ionomycin. Cells were stimulated for 4 h with lipopolysaccharide, γIFN, or lipopolysaccharide plus γIFN in the absence (lanes 2, 4, 6, 8) or presence (lanes 1, 3, 5, 7) of 1 μM ionomycin and incubated with 2 MAC isoflurane (negative controls, lanes 7 and 8). Similar results were obtained in three separate experiments. (C ) iNOS mRNA levels of immunostimulated cells from three separate experiments were quantified using a phosphor imager. Cells were stimulated and incubated with 2 MAC isoflurane either in the absence of ionomycin (filled bars) or in the presence of ionomycin (stippled bars). The effect of ionomycin on immunostimulated iNOS transcription in cells not incubated with isoflurane was also shown (hatched bars). Values are expressed as the percentage (mean ± SEM) of the corresponding controls (100%) that were immunostimulated in the absence of isoflurane and ionomycin (open bars). *P < 0.05 compared with the corresponding controls by Mann–Whitney U test.