Cloning and characterization of a novel form of mouse fibroblast growth factor-1 (FGF-1) mRNA, FGF-1.G: differential expression of FGF-1 and FGF-1.G mRNAs during embryonic development and in postnatal tissues.

The fibroblast growth factor-1 (FGF-1) gene is characterized by the presence of different untranslated exons in its 5' end that direct the expression of alternatively spliced mRNA variants (1.A, 1.B and 1.C) that encode for FGF-1. We have previously isolated a new mouse FGF-1 upstream untranslated exon, which we termed -1G. Here we report on the cloning and characterization of the FGF-1 mRNA isoform arising from -1G. This newly identified FGF-1 mRNA species (FGF-1.G), whose transcription start site maps 295 bp upstream from the splice donor site, is predominantly expressed in young liver and kidney, where it comprises 40.2% and 30.7%, respectively, of the total FGF-1 mRNA. While the FGF-1 mRNA comprising all of the FGF-1 transcripts was present in distinct tissues at embryonic days E12.5 and E15.5, the FGF-1.G mRNA was not detected during murine embryogenesis; therefore the role of FGF-1 in embryonic development must be attributed to FGF-1 mRNAs arising from upstream untranslated exons other than -1G. On the other hand, the parallel decrease of both FGF-1 and FGF-1.G mRNA levels we observed in the aging mouse kidney and liver suggests a role of FGF-1.G in normal cellular maintenance and survival.

Lack of FGF-1 overexpression during autoimmune nephritis in the kidneys of MRL lpr/lpr mice.

Fibroblast growth factors (FGFs) have been implicated in the pathobiology of autoimmune inflammation on the basis of their angiogenic properties, as well as their increased expression at inflammatory sites. In order to better evaluate the role of FGF-1 in the renal disease of mice that develop spontaneous lupus-like clinical features, we examined the expression of FGF-1 mRNA and protein in the kidneys of the murine MRL lpr/lpr strain. Both Northern and Western blot analyses demonstrated that FGF-1 levels do not increase in the kidneys of this particular autoimmune mouse strain as a function of renal disease. These results suggest that FGF-1 may not be involved in the pathogenesis of autoimmune nephritis in MRL lpr/lpr mice.

Characterization of the entire transcription unit of the mouse fibroblast growth factor 1 (FGF-1) gene. Tissue-specific expression of the FGF-1.A mRNA.

Fibroblast growth factor 1 (FGF-1, also known as acidic FGF) is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells, as well as an angiogenic factor in vivo. It has been implicated in angiogenic diseases including atherosclerosis, cancer and inflammatory diseases. In the present study, the entire transcriptional unit of the mouse FGF-1 gene, including four promoters, is characterized. By nucleotide sequence and RNase protection analyses, we have determined that its 3'-end resides 3.2 kilobase pairs downstream from the stop codon. We have previously cloned and characterized the mouse homologue of the human 1B promoter, as well as a novel upstream untranslated exon. In order to elucidate the regulatory mechanism of FGF-1 gene expression, the mouse promoter containing TATA and CAAT consensus sequences (FGF-1. A) was isolated from a P1 library and characterized. We further determined that the mouse heart is the most abundant source for the FGF-1.A mRNA. Finally, via both RNase protection analysis and 5'-rapid amplification of cDNA ends, we determined the transcription start site of the FGF-1.A mRNA.

Downregulation of class II antigen expression by FGF-heparin complexes is due primarily to heparin effect.

Fibroblast growth factors are thought to play a role in the pathogenesis of autoimmune inflammation. The evidence linking these growth factors to autoimmunity stems in part from their presence in mononuclear cells from inflammatory sites during disease processes. We sought to further dissect the mechanisms through which fibroblast growth factors might affect the inflammatory response. Peritoneal macrophages from autoimmune MRL 1pr/1pr mice and congenic wild-type MRL +/+ mice were cultured for 72 hours in the presence of either IFN-gamma, heparin, FGF-1, FGF-2, FGF-1 plus heparin, FGF-2 plus heparin or medium alone. Expression of MHC class II (I-Ak and I-Ek) antigens were analyzed using direct immunofluorescence and flow cytometry. As expected, at baseline there were higher numbers of I-Ak bearing cells in elicited peritoneal cells from 1pr mice relative to +/+ cells (70.8 +/- 14.9 versus 43.4 +/- 19.7, p = 0.046). Expression of I-Ak and I-Ek and percentage of I-E bearing cells were essentially the same between strains. Cells from 1pr and +/+ mice displayed reductions in the percentage of I-Ak expressing cells following culture with FGF-1 plus heparin and FGF-2 plus heparin. Similarly, cells from both 1pr and +/+ mice displayed significant reductions from baseline I-Ak expression following culture with FGF-1 and FGF-2 in the presence of heparin. Similar reductions were seen in cells from both strains cultured with heparin alone. No change from baseline was discernible when cells were cultured in the presence of FGF-1 or FGF-2 alone. Titration studies showed a maximum heparin effect at 5 units/ml culture. However, reduced amounts of heparin in the cell culture were directly proportional to decreased levels of I-Ak expression. These results suggest that cells from autoimmune MRL 1pr/1pr mice and wild type MRL +/+ mice respond similarly with a general reduction of I-Ak expression and a decrease in the percentage of I-Ak bearing cells in response to heparin with little discernible effect from addition of either FGF-1 or FGF-2. This change in class II expression suggests that the heparin-heparan component in FGF-heparin complexes may serve to downregulate class II expression during inflammation.

Immunologic profile of patients with fibromyalgia.

Fibromyalgia is a musculoskeletal disorder characterized by generalized myalgias, arthralgias widespread tender points in discreet areas on examination. It is frequently accompanied by fatigue, stiffness, and a nonrestorative sleep pattern. These patients generally have a normal blood count and chemistry profile. There is a subset of people with fibromyalgia (FM) who test positive for the antinuclear antibody (ANA) and have constitutional symptoms that resemble those of patients with early lupus. We studied the immunologic profile of patients with FM who are ANA-positive (+). A retrospective review of patient records in a university-based rheumatology practice was conducted. In a group of 66 FM patients, 30% (20) were ANA+, with a 75% preponderance of the speckled pattern and 20% diffuse pattern. The remaining 5% were equally split between diffuse-speckled and speckled-nucleolar patterns. All had negative staining for extractable nuclear antibodies. The Smart Index (SI), a ratio of the sedimentation rate to one-half the patient's age, was developed to characterize each patient's inflammatory response. The FM patients who were ANA negative (-) had a mean SI of 0.55, whereas the FM patient's who were ANA+ had a SI of 1.07. These ANA+ patients represent a subgroup of patients who have FM with an inflammatory response profile larger than that of the ANA-patients.

Gallium nitrate suppresses lupus in MRL/lpr mice.

Gallium (Ga) nitrate, a drug which prevents a variety of experimental autoimmune diseases, was investigated in a murine model of systemic lupus erythematosus (SLE). In one experiment, female MRL/Mp lpr/lpr (MRL/lpr) mice were randomized into 2 groups of 6: 1) vehicle (trisodium citrate) and 2) Ga. Subcutaneous injections began at 3 weeks of age and continued weekly until the mice were euthanized a week after the thirteenth injection. The loading dose of Ga (calculated as elemental Ga) was 45 mg/kg, followed by 15 mg/kg/week. In another experiment (n = 18) with 3 males and 3 females per group, mice received 1) vehicle, 2) Ga x 1 (one 45 mg/kg dose), and 3) Ga x 13. In the experiment with 12 mice, axillary lymph nodes from Ga-treated mice were significantly smaller than those from vehicle-treated mice (91+/-42 and 360+/-358 mg respectively, mean+/-SD), and spleens as well as lymph nodes from the former showed significantly less lymphoid infiltrate. In the experiment with 18 mice, prescapular lymph nodes weighed 312+/-98, 217+/-52, and 42+/-34 mg, and spleens weighed 732+/-492, 409+/-164, and 192+/-93 mg in the groups which received vehicle, Ga x 1, and Ga x 13 respectively. Control mice had significantly more lymphoid infiltrates in the lungs, spleen, and lymph nodes and, unlike Ga x 13 mice, exhibited glomerulitis and renal vasculitis. Within groups, females developed more severe disease than males. The Ga x 13 group had increased percentages of CD4-bearing and CD8-bearing lymphocytes in lymph nodes and increased CD4-bearing lymphocytes in the spleen, with an increased proliferative response to mitogen stimulation in vitro in lymph nodes, although not in the spleen. The Ga x 13 group also gained less weight and developed osteosclerosis. Although preliminary, our findings suggest that clinical trials with Ga in SLE are merited.

Characterization of the 1B promoter of fibroblast growth factor 1 and its expression in the adult and developing mouse brain.

The present study elucidates the molecular structure of a murine fibroblast growth factor 1 (FGF-1) promoter and describes its distribution in the adult and developing mouse brain. A cDNA clone coding for FGF-1 was isolated from a mouse brain cDNA library. Nucleotide sequence analysis revealed that the clone contained, in addition to the protein coding region, an untranslated exon (FGF-1B) 34 base pairs upstream of the translation start codon ATG. The mouse cDNA clone corresponded to the sole FGF-1 transcript in the brain. An RNase protection assay was used to map the transcription start site of the 1B promoter. The sequences upstream from the major transcription initiation site lacked consensus TATA or CAAT boxes. In situ hybridization with cRNA probes specific for the 1B transcript showed the message to be restricted largely to sensory and motor nuclei in the brainstem, and to the ventral spinal cord and cerebellum. Although occasional brainstem nuclei were labeled at low levels by embryonic day 18, the majority of nuclei became detectable autoradiographically during postnatal weeks 1 and 2, and adult levels of grain density were reached during the 3rd and 4th postnatal weeks. FGF-1B mRNA was expressed in phylogenetically older brain regions, which are involved primarily in processing information from exteroceptive sensory mechanoreceptors and in motor control. The relatively late developmental expression suggests a role for FGF-1 in neuronal maturation, rather than in neurogenesis.

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene.

Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5'-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.

Cloning and characterization of the mouse Fgf-1 gene.

Fibroblast growth factor 1 (FGF-1 or aFGF), is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells, as well as an angiogenic factor in vivo. It has been implicated in angiogenic diseases including atherosclerosis, cancer and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the pathobiology of inflammation, we have isolated and characterized the mouse Fgf-1 gene. Southern blot analysis of mouse genomic DNA using the mouse Fgf-1 cDNA as a probe revealed that mouse FGF-1 is encoded by a single copy gene. Comparison of the available mouse Fgf-1 cDNA sequence with newly obtained genomic sequence allowed us to establish the exon/intron boundaries. The mouse Fgf-1 coding region is comprised of three protein coding exons, which we determined to be separated by an 11.4-kb and a 4.9-kb intron. The elucidation of the mouse Fgf-1 coding region revealed great similarity between the mouse and human Fgf-1 gene structure.

Acidic and basic fibroblast growth factors prolong the in-vitro survival of murine peritoneal macrophages.

To examine the role of fibroblast growth factors on the proliferation present during angiogenic processes, we analyzed the effects of in vitro cell culture with acidic (FGF-1) and basic (FGF-2) fibroblast growth factors on murine peritoneal exudate cell survival. FGF-1 or FGF-2 in the presence of heparin resulted in a significantly greater cell viability for exudate cells at 24 hours of culture relative to heparin alone or medium controls. Cultures supplemented with FGF-1 or FGF-2 plus heparin showed a slight increase in tritiated[3H] thymidine uptake over heparin or medium alone. No specific change in [3H] uridine incorporation or LDH release was detectable between FGF-1, FGF-2, heparin or medium alone cultures. Our results suggest that FGF-1 and FGF-2 may enhance survival both in vitro and in vivo of a subset of peritoneal exudate cells. This attribute may play a role during inflammatory or tumor-like states.

Environmental influences on fatty acid composition of membranes from autoimmune MRL lpr/lpr mice.

We analyzed fatty acid make up of cells and organs from autoimmune and immunologically normal mice to determine whether intrinsic differences in composition might be associated with an inflammatory phenotype. Macrophages (MO) isolated from 4-6-week-old MRL lpr/lpr mice were cultured with phorbol ester (PMA), fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2) and medium control to determine whether these cell signals might induce membrane fatty acid changes. Individual phospholipid analysis showed 8.4- and 5.1-fold increases in phosphatidylcholine arachidonate (20:4) mole % over baseline values following culture with FGF-1 and FGF-2, respectively. Unfractionated analysis on kidney and liver extracts from 4-6 week MRL lpr/lpr, 16-20 week lpr and 12-20 week MRL +/-/+/- mice demonstrated no significant intrastrain fatty acid differences. Higher levels of 20:4 in 4-6 week lpr mice were noted compared to 16-20 week lpr or +/+ mice in kidney, and liver samples (P < 0.05). It is possible that membrane changes precipitated by microenvironmental cytokine concentrations may contribute to the expression of autoimmune disease in this model.

Calcium signals and protein tyrosine kinases are required for the induction of c-jun in Jurkat cells stimulated by the T cell-receptor complex and oxidative signals.

The regulation of c-jun plays an important role in T cell activation, proliferation, and expression of interleukin-2. In the present study, we determined whether Ca2+ signals and the activity of protein tyrosine kinases (PTKs) were required for the induction of c-jun in Jurkat cells stimulated with cross-linked anti-T cell receptor/CD3 antibodies or exposed to oxidative stress in the form of micromolar concentrations of H2O2. Jurkat cells exhibited rapid elevations in intracellular calcium [Ca2+]i levels in response to H2O2 and cross-linked anti-CD3 antibodies that mainly reflected the influx of extracellular Ca2+. The Ca2+ flux in response to oxidative signals was distinguished by an exquisite sensitivity to inhibition with Ni2+, suggesting the involvement of cation channels. PTK activity was needed for [Ca2+]i elevations in response to both oxidative and anti-CD3 signals, although H2O2 induction of [Ca2+]i increases was more resistant to inhibition by genistein than anti-CD3 [Ca2+]i responses. Both oxidative signals and anti-CD3 stimulation induced increased levels of c-jun and c-fos mRNA. The increased expression of c-jun with H2O2 was preceded by [Ca2+]i increases and accompanied by activation of c-Jun aminoterminal kinases (JNKs), as well as increased AP-1 binding activity. Induction of c-jun with oxidative signals and anti-CD3 was also shown to be crucially dependent on [Ca2+]i elevations because the chelation of [Ca2+]i with BAPTA resulted in a dose-dependent inhibition of c-jun expression. Furthermore, inhibition studies demonstrated that the optimal induction of c-jun mRNA in response to oxidative signals required PTK as well as protein kinase C (PKC). Thus, these findings suggest that both [Ca2+]i signals and the activity of PTKs are essential for the optimal expression of c-jun in response to TCR/CD3 signals and changes in redox potentials.

A pilot study of zileuton, a novel selective 5-lipoxygenase inhibitor, in patients with systemic lupus erythematosus.

OBJECTIVE: This is a pilot study of zileuton, a selective 5-lipoxygenase inhibitor in systemic lupus erythematosus (SLE). METHODS: Forty patients with SLE received zileuton 600 mg qid or placebo in an 8 week, randomized prospective, double blind trial. Disease activity was manifested largely by constitutional, articular, and skin manifestations with no evidence of active renal, cardiac, or neurologic involvement. Concomitant administration of nonsteroidal antiinflammatories, corticosteroids, or antimalarials was not permitted. Disease activity was determined at baseline and at Days 15 and 57 by assessment of arthritis severity, the Systemic Lupus Activity Measure (SLAM), investigator and patient global ratings, hematologic indices, and serologic measures including autoantibody titers, complement levels, and interleukin 2 receptors (IL-2R). Total body sulfidopeptide leukotriene synthesis was measured by urinary leukotriene E4 (LTE4) concentrations. RESULTS: Overall SLAM (the primary measure of efficacy in this study) was significantly improved with zileuton compared with placebo (-2.1 +/- 1.3, compared with an increase of 2.3 +/- 1.3 with placebo by Day 57, p = 0.048). Changes in individual SLAM subscores, arthritis severity, global ratings, and IL-2R levels compared with baseline did not achieve statistical significance, but were generally decreased from baseline with zileuton (indicating trends towards improvement) and increased from baseline with placebo (indicating trends towards clinical worsening). Urine LTE4 levels at Day 57 had increased from baseline in the placebo group (indicating worsening) and decreased in the zileuton group (indicating improvement). CONCLUSION: Selective 5-lipoxygenase inhibition may be beneficial in mild SLE.

Differential expression of the alpha- and beta-isoforms of protein kinase C in peripheral blood T and B cells from young and elderly adults.

The expression of alpha- and beta-isoforms of protein kinase C (PKC) was analyzed in the peripheral blood T and B cells from 11 elderly and young humans. Immunoblot analysis with isoenzyme specific antibodies showed that T cells from five of 11 elderly subjects exhibited selective reductions in PKC alpha which was < 60% of those in young subjects whereas the levels of PKC beta were comparable to T cells of young subjects. No age-related reductions of PKC alpha or beta were observed in B cells. Among individual elderly subjects, the reductions in T cell PKC alpha were not associated with lower levels of PKC beta thereby resulting in only approximately 60-70% reductions of combined PKC alpha plus PKC beta. In addition, the functional properties of PKC in stimulated T cells of elderly subjects with respect to activation/translocation were comparable to T cells of young subjects. These results suggest that selective alterations in PKC isoenzymes can occur in human T cells during aging which may not be readily apparent in standard enzymatic assays and may contribute to aberrancies in intracellular signal transduction.

Regulation of protein kinase enzymatic activity in Jurkat T cells during oxidative stress uncoupled from protein tyrosine kinases: role of oxidative changes in protein kinase activation requirements and generation of second messengers.

Prior studies have suggested that intracellular phosphorylation events and cellular redox mechanisms may interact in regulating a variety of cellular functions, including the transcriptional activation of gene expression. Increased activity of transcriptional factors NF kappa B and AP1 has been described in cells exposed to oxidative stress and following the direct stimulation of protein kinase C (PKC) by phorbol diesters. However, the mechanisms that may contribute to redox regulation of PKC are unknown. We studied the expression of PKC activity and several second messengers in human Jurkat T cells exposed to oxidative stress in the form of H2O2. Micromolar concentrations of H2O2 rapidly induced increased cytosolic PKC enzymatic activity in Jurkat T cells that was associated with a marked arrest of cellular proliferation. The increase in cytosolic PKC activity in cells treated with H2O2 was accompanied by elevations in intracellular free calcium ([Ca2+]i), generation of inositol phosphates, and release of arachidonic acid. Functional studies showed that H2O2 enhancement of cytosolic PKC activity required phospholipase C activity but was not primarily mediated by arachidonic acid. The response of PKC to oxidative stress displayed a lack of Ca2+ dependence and was uncoupled from the activity of protein tyrosine kinases (PTK). Furthermore, the reduced activation requirements of PKC from cells treated with H2O2 were associated with shifts in elution profiles of PKC enzymatic activity after Mono-Q chromatography. These shifts appeared to represent intrinsic changes in the conformation of PKC induced by oxidative stress because western blotting failed to reveal any PKC cleavage products or reductions in native PKC alpha or beta. These findings indicate that oxidative regulation of intracellular events can intersect phosphorylation events mediated by PKC through the release of second messengers as well as direct changes in PKC activation requirements. Moreover, redox regulation of PKC is distinct from T cell receptor signaling in that the activity of PKC is uncoupled from the regulatory influences of PTK.

Composition of peritoneal macrophage membranes in autoimmune MRL lpr/lpr mice.

Adult MRL lpr/lpr mice display phenotypic features that are consistent with both rheumatoid arthritis and systemic lupus erythematosus. Previous studies have reported that peritoneal macrophages harvested from this model have an increased propensity for both spontaneous and elicited release of prostaglandins and leukotrienes relative to immunologically normal control mice. To investigate whether one aspect of the differences in secretory potential between autoimmune and normal mice was at the level of increased substrate availability, gas chromatographic analysis of peritoneal macrophage membranes from autoimmune MRL lpr/lpr, young lpr, wild type +/+, and immunologically normal mice was done. The results demonstrate enrichment of arachidonate in adult lpr macrophage membranes in all major phospholipid classes relative to young lpr, +/+ and immunologically normal C3H/HeN mice. Similarly, there was an increased mole % of arachidonic acid in lpr mice relative to controls. Elevated membrane arachidonate may contribute to the increased propensity of autoimmune strains to participate in the inflammatory process.

Fibroblast growth factors mobilize peritoneal macrophage intracellular calcium.

Macrophages have been implicated in the propagation of inflammatory disease. The evidence linking macrophages to inflammation stems from their elicited responses to various extracellular ligands eventually culminating in the elaboration of a variety of inflammatory mediators. As part of an investigation of fibroblast growth factors role in promoting inflammation, we examined one aspect of transmembrane signal transduction, intracellular calcium mobilization following culture of murine peritoneal macrophages with acidic and basic fibroblast growth factor. Peritoneal macrophages displayed a rapid rise in cytosolic calcium from a basal level of 147.6 +/- 25.4 nM to 261.9 +/- 49.9 nM at 3.5 minutes following culture with acidic fibroblast growth. A similar rise in calcium was noted with basic fibroblast growth factor. Titration revealed the maximal effective dose of aFGF and bFGF with respect to calcium response to be 10 ng/ml. Using blockers of both voltage and non-voltage gated channels, the FGF induced rise in cytosolic calcium was specifically abolished. Similarly, using specific 5-lipoxygenase (A69412) or cyclooxygenase (Indomethacin) blockers, the aFGF induced rise in maximal calcium response was reduced by 41% and 96% respectively. On the basis of these data, we speculate on some possible roles that FGF may play in the inflammatory response.

Urine leukotriene E4 levels are elevated in patients with active systemic lupus erythematosus.

The peptidoleukotrienes, leukotriene (LT) C4 and its metabolites LTD4 and LTE4, cause diverse physiologic effects and have been implicated in several disease processes. A potential role for enhanced peptidoleukotriene synthesis in the pathogenesis of autoimmune disease in general and systemic lupus erythematosus (SLE) in particular has been suggested by animal studies. Therefore, we measured the urinary levels of LTE4 in patients with active and inactive SLE as well as in patients with rheumatoid arthritis (RA), scleroderma (Scl), and in healthy controls. Comparisons were made to other standard clinical tests in assessing individual patient disease activity. A marked increase in urinary LTE4 levels in patients with active SLE was noted (319 +/- 49 pg/mg creatinine, n = 20) relative to patients with inactive SLE (80 +/- 8 pg/mg creatinine, n = 7 [p less than 0.02]), patients with RA (86 +/- 8 pg/mg creatinine [p less than 0.01]), and healthy controls (68 +/- 4.3 pg/mg creatinine, n = 6 [p less than 0.01]). Patients with Scl also had elevated urinary LTE4 levels (188 +/- 33 pg/mg creatinine, n = 7) relative to controls (p less than 0.02), while values from patients with RA were not significantly different from controls. Using the Systemic Lupus Activity Measurement as a gauge of clinical activity, a rise in urinary LTE4 levels was noted during stages of active disease with a subsequent decline following the resolution of these symptoms. Our data indicate that increased synthesis of leukotrienes is associated with active SLE and Scl and suggest that these leukotrienes may mediate certain symptoms associated with these diseases.

Naloxone in septic shock.

Treatment of septic shock is a persistent dilemma. The clinical use of agents such as naloxone has resulted in variable success. Because the dosage and timing of these agents are considered critical factors in their efficacy, we investigated both dosage and timing of naloxone. Thirteen consecutive patients with documented septic shock and resistance to a one-liter fluid challenge underwent invasive hemodynamic monitoring and the administration of naloxone by initial bolus of 0.03 mg/kg followed by infusion at a rate of 0.2 mg/kg.h over one hour. During the one-hour observation period, iv fluid administration, concomitant pressor agents, and respirator values were constant. After infusion, adjustments in fluid administration, respirator status, and pressor agents were made as required by the clinical situation. A significant increase in mean arterial pressure (MAP) over baseline (60 +/- 3 mm Hg) was noted at 5 min (77 +/- 6 mm Hg, p less than .005) and at 30 min (73 +/- 6 mm Hg, p less than .025). Similarly, a significant increase in systolic arterial pressure was noted over prenaloxone levels (89 +/- 3 mm Hg) at 5 min (114 +/- 6 mm Hg, p less than .001), 30 min (107 +/- 8 mm Hg, p less than .05), and at one hour (106 +/- 8 mm Hg, p less than .05). There was a moderate nonsignificant increase in cardiac index, pulmonary capillary wedge pressure, and systemic vascular resistance. No side-effects to naloxone were noted in our group. No effect on survival could be demonstrated. We found no overall effect on mortality. However, by its increase of MAP, naloxone may serve as a temporizing agent during the treatment of critically ill patients with septic shock.