[In vitro activity of ertapenem against clinical bacterial isolates in 69 Spanish medical centers (E-test study)]. / Actividad in vitro de ertapenem frente a cepas bacterianas clínicas aisladas en 69 centros médicos españoles (Estudio E-test).

This study was conducted to assess the in vitro activity of ertapenem against clinical bacterial isolates from patients with community-acquired intra-abdominal and lower tract respiratory infections in Spain in 2003. As the study was conducted before the marketing of ertapenem, it was also useful to define a baseline susceptibility pattern for ertapenem in each of the participating hospitals for later surveillance studies. Each partipating site identified a variable number of aerobic and facultative bacteria isolated from patients with community-acquired intra-abdominal infection or pneumonia using standard procedures. E-test strips were used for determining the minimum inhibitory concentration (MIC) of ertapenem, while for other antimicrobials either quantitative dilution techniques or qualitative diffusion procedures were used according to each microbiology laboratory's routine practice. MIC breakpoints for categorization of susceptibility provided by the CLSI were used for interpreting MIC values. A total of 2,901 recent clinical isolates from patients with community-acquired intra-abdominal infection or pneumonia hospitalized in 69 Spanish medical centers were tested. These isolates included 2,039 Gram-negative bacteria (1,646 Enterobacteriaceae, 216 Haemophilus, 123 non-fermenting Gram-negative bacteria [NFGNB] and 54 others) and 862 Gram-positive bacteria (556 pneumococci, 159 staphylococci, 96 streptococci other than S. pneumoniae, 44 enterococci and 7 others). Ertapenem was very active in vitro against Enterobacteriaceae (99.8% susceptible), Haemophilus (96.3% susceptible), pneumococci (99.6% susceptible, of which 31% were penicillin non-susceptible strains), streptococci other than S. pneumoniae (99.0% susceptible) and methicillin-susceptible staphylococci (94.8% susceptible). For other Gram-positive and Gram-negative pathogens for which ertapenem susceptible breakpoints have not been defined, MIC(90) values were 0.38 and 0.064 mg/l, respectively. As expected, ertapenem had minimal activity in vitro against NFGNB, enterococci and methicillin-resistant staphylococci (MIC(90) of >32 mg/l for all three). Ertapenem was highly active in vitro against most bacteria isolated from patients with community-acquired intra-abdominal and lower respiratory tract infections.

Inhibition of Trypanosoma cruzi hexokinase by bisphosphonates.

Hexokinase is the first enzyme involved in glycolysis in most organisms, including the etiological agents of Chagas disease (Trypanosoma cruzi) and African sleeping sickness (Trypanosoma brucei). The T. cruzi enzyme is unusual since, unlike the human enzyme, it is inhibited by inorganic diphosphate (PPi). Here, we show that non-hydrolyzable analogues of PPi, bisphosphonates, are potent inhibitors of T. cruzi hexokinase (TcHK). We determined the activity of 42 bisphosphonates against TcHK, and the IC(50) values were used to construct pharmacophore and comparative molecular similarity indices analysis (CoMSIA) models for enzyme inhibition. Both models revealed the importance of electrostatic, hydrophobic, and steric interactions, and the IC(50) values for 17 active compounds were predicted with an average error of 2.4x by using the CoMSIA models. The compound most active against T. cruzi hexokinase was found to have a 2.2 microM IC(50) versus the clinically relevant intracellular amastigote form of T. cruzi, but only a approximately 1-2 mM IC(50) versus Dictyostelium discoideum and a human cell line, indicating selective activity versus T. cruzi.

[Patterns of susceptibility to antibiotics of Enterobacteriaceae causing intra-abdominal infection in Spain: SMART 2003 study outcomes]. / Patrones de sensibilidad a antimicrobianos de Enterobacteriaceae causantes de infecciones intraabdominales en España: resultados del estudio SMART 2003.

SMART (Study for Monitoring Antimicrobial Resistance Trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intra-abdominal infections. The objective of this subanalysis was to assess antimicrobial susceptibility patterns among Entero-bacteriaceae recovered at 13 participating Spanish sites during 2003. Antimicrobial susceptibility testing was performed using broth microdilution techniques according to the CLSI (formerly NCCLS) guidelines for MIC testing. The presence of extended-spectrum beta-lactamases (ESBL) was confirmed in isolates with a MIC of ceftriaxone, ceftazidime, or cefepime>or=2 mg/l by comparing cefepime MICs with and with-out clavulanate. A total of 981 Enterobacteriaceae recovered from 840 patients were tested, of which 398 (41%) were community-acquired. Escherichia coli was the most common isolate (571 isolates; 58%), followed by Klebsiella spp. (153; 16% Enterobacter spp. (97; 10%), and Proteus spp. (63; 6%). A total of 191 isolates (19%) from 176 patients produced inducible beta-lactamases. The carbapenems and amikacin were the most consistently active agents against the Enterobacteriaceae (susceptibility>or=99%). Resistance rates for ceftazidime, cipro-floxacin, and levofloxacin exceeded 10%. ESBLs were detected phenotypically in 61 (6%) isolates, being the most common E. coli (61%), Klebsiella spp. (20%), and Enterobacter spp. (8%). Antimicrobial resistance among Enterobacteriaceae isolated from intra-abdominal infections is a problem in Spain. A significant proportion of inducible beta-lactamase and ESBL-producing Enterobacteriaceae causing intra-abdominal infection were acquired in the community. The carbapenems ertapenem, imipenem and meropenem and the aminoglycoside amikacin were highly active in vitro against Enterobacteriaceae isolated from intra-abdominal sites, including ESBL-producing organisms.

Successful treatment of rhinocerebral zygomycosis with a combination of caspofungin and liposomal amphotericin B.

Genera of the order Mucorales (Rhizopus, Mucor, Rhizomucor, Absidia, Apophysomyces, Cunninghamella, and Saksenaea) cause an angioinvasive infection called zygomycosis. Mortality rates can approach 100% depending on the patient's underlying disease and form of zygomycosis. We report here on the unusual case of a patient with acute myelogenous leukemia and zygomycosis unresponsive to monotherapy with liposomal amphotericin B, who responded favorably following the addition of the echinocandin caspofungin acetate.

Long-term management of homozygous protein C deficiency: replacement therapy with subcutaneous purified protein C concentrate.

We present the case of a full-term newborn in whom purpura fulminans developed shortly after birth. A diagnosis of homozygous protein C deficiency was established based upon undetectable plasma protein C activity and antigenemia in the newborn infant, and was later confirmed by protein C gene analysis. Specific replacement therapy with intravenous protein C concentrate was started 9 days after birth. This rapidly led to the complete regression of cutaneous lesions and consumption coagulopathy. After stabilization, oral anticoagulation was initiated in association with prophylactic treatment with intravenous protein C concentrate. However, oral anticoagulation was finally abandoned as the patient presented several thrombotic and hemorrhagic episodes clearly related to difficulties with anticoagulation. Due to the hazards related to prolonged venous access, we are currently using subcutaneous infusion of protein C concentrate for the long-term management of this condition, with satisfactory results.

Plasma endothelin-1 levels after stem cell transplantation.

Acute renal failure and veno-occlusive disease of the liver are serious complications following stem cell transplantation (SCT) and contribute to the non-relapse mortality associated with this procedure. Endothelins, a family of vasoconstrictor peptides, may be involved in the pathogenesis of a variety of renal and hepatic diseases, including CsA-associated hypertension and the hepatorenal syndrome. In order to study the relevance of endothelins to SCT-related liver and kidney dysfunction, we determined endothelin-1 (ET-1) levels in plasma samples obtained from 65 patients (38 autologous, 27 allogeneic) 7 days before and 7, 14 and 28 days after SCT. A steady increase in plasma ET-1 was observed after SCT (5.36 pg/ml, 95% CI 4.30-6.43 on day +28 vs 3.82 pg/ml, 95% CI 3.21-4.43 on day -7; P = 0.020). No differences in ET-1 levels existed between autologous and allogeneic SCT recipients at any of the time points studied (P = 0.561). In addition, no significant differences were observed among patients with renal dysfunction vs those without (P = 0.187), nor in patient groups with or without hepatic dysfunction (P = 0.075). In conclusion, even though plasma ET-1 levels showed a steady increase following SCT, no correlation could be found with development of SCT-related kidney or liver dysfunction.

Safety of the concomitant use of caspofungin and cyclosporin A in patients with invasive fungal infections.

Caspofungin, an echinocandin antifungal agent, is active against invasive Aspergillus and Candida infections. In a phase I study in healthy volunteers, mild transient increases in serum aminotransferases were observed with the concomitant administration of caspofungin and cyclosporin A (CsA). As a result, it is recommended that the concomitant use of the two drugs be limited to those settings with appropriate risk-benefit balance. We retrospectively assessed safety data in 14 patients with refractory invasive mycoses who were treated concomitantly with CsA and caspofungin before the drug was licensed in Spain. In all, 13 patients were adults (median age, 31.5 years; range, 14-67 years). The average duration of concomitant therapy was 15 days (range, 2-43 days). No clinically significant elevations of serum aminotransferases were observed, and no patient had concomitant therapy discontinued or interrupted due to a drug-related adverse event. In this study of a limited number of patients, the coadministration of caspofungin and CsA was generally well tolerated.

Interferon alpha for chronic myeloid leukemia relapsing after allogeneic bone marrow transplantation.

Interferon alpha (IFN alpha) induces cytogenetic responses in patients with chronic myeloid leukemia (CML) who relapse after allogeneic bone marrow transplantation (BMT). The purpose of this study was to analyze the therapeutic role of IFN alpha in this setting. The experience of a single institution and the published results on this topic were evaluated. We have included patients who received IFN alpha as a single agent, excluding those patients who received previous or simultaneous donor leukocyte infusions. The outcomes of 11 patients treated in our center and those of 108 previously reported patients have been analyzed. Five out of 11 patients treated in our institution obtained a complete cytogenetic response (CGR). Two patients continue in complete cytogenetic response 3.5 and 8.2 years later, and the qualitative RT-PCR is negative for bcr-abl RNA. The CGR has been transient in one patient, and follow-up is short in the other two. Secondary effects have been acceptable, with myelosuppression as the main toxic effect. Graft-versus-host disease did not occur. The literature review identified 108 patients treated with IFN alpha as sole therapy for relapsed CML. Cytogenetic response and CGR seem to be better in patients with cytogenetic relapse, as compared to patients with hematologic relapse (61% vs. 45% and 45% vs. 28%, respectively). Several patients remained in CGR for more than 5 years. This overview also suggests that CGR is more frequent when IFN alpha is used in patients relapsing after non T-depleted BMT. IFN alpha induces complete cytogenetic response in nearly half of the patients with CML who relapse after allogeneic BMT, with acceptable toxicity. We believe that these results using IFN alpha as a front-line therapy for CML relapsing after BMT warrant a randomized comparison with donor lymphocyte infusions.

Cytosine arabinoside is neurotoxic to chick embryo spinal cord motoneurons in culture.

Cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, AraC) is a commonly used antimitotic agent that kills proliferating cells by inhibiting DNA synthesis. We report that AraC is toxic to cultured chick embryo spinal cord motoneurons (MTNs) in a concentration-dependent fashion with an EC50 of about 2 microM. Interestingly, this type of MTN death is specific, resembles that occurring upon muscle extract (MEX) trophic deprivation regarding its morphological and temporal characteristics, and has apoptotic features, as judged by observation of nuclear morphology. The death of AraC-treated MTNs can be blocked by 2'-deoxycytidine (dC), a pyrimidine metabolite AraC is structurally related to. Overall, these findings suggest that dC may participate in a pathway, different from inhibition of DNA synthesis, that is necessary for cultured MTNs to respond to the trophic activities present in MEX.

[Hematopoietic stem cell transplantation as salvage treatment in patients with aggressive non-Hodgkin's lymphoma]. / Trasplante de células progenitoras hematopoyéticas como tratamiento de rescate en pacientes con linfomas no hodgkinianos agresivos.

BACKGROUND: The indication of early hematopoietic stem cell transplantation (HSCT) in patients with aggressive non-Hodgkin's lymphoma (LNH) is controversial. PATIENTS AND METHODS: Retrospective analysis of 86 patients with aggressive NHL treated with MACOP/VACOP-B chemotherapy. HSCT was performed as salvage treatment to patients under 65 years of age with progressive disease or chemosensitive relapse. Progression free survival (PFS) and overall survival (OS) were determined by the Kaplan-Meier method. Rates of response and survival functions were compared between the International Prognostic Index (IPI) groups using the Chi-square and log-rank tests, respectively. RESULTS: Patients median age was 48 years; 22% had T cell NHL and 57% had intermediate-high and high risk (high risk) IPI. There were 6 toxic deaths (7%), and treatment failure was observed in 42 patients (48.8%). Thirty one of them were candidates for TPH due to age under 65 years, although 21 were finally transplanted (including 13 with high risk IPI). A significant association between PFS and IPI was observed, 61.9% for low risk (low and low-intermediate) versus 28.2% for high risk groups (p = 0.0007). With a median follow up of 4.8 years, OS was 64%; 80.5% for low risk versus 52.6% for high risk IPI groups (p = 0.01), and 83.7% versus 62% for the same groups in patients under 65 years of age (p = 0.02). The median follow up after failure to chemotherapy was 42.7 months. CONCLUSIONS: In this retrospective study, OS rate in high risk IPI patients with NHL using HSCT as salvage treatment is similar to that reported using HSCT during earlier phases of treatment.

Expression of Fc gamma receptor-mediated phagocytosis by airway intra-luminal macrophages.

Macrophages in the conducting airways of the lower respiratory tract constitute an anatomically defineable subpopulation of pulmonary macrophages. Little information regarding the functional characteristics of the airway intra-luminal macrophages (AI-LM) is currently available. In this study, the AI-LM resident in the trachea and mainstem bronchi of the rat were harvested by an airway lavage technique and the ability of the AI-LM to phagocytize by Fc gamma-receptors was evaluated relative to the phagocytic activities of alveolar macrophages (AM) obtained by bronchoalveolar lavage. More than 60% of the AI-LM phagocytized sheep erythrocytes opsonized with IgG (SRBC-IgG) and the distributions of the engulfed SRBC-IgG in the phagocytic AI-LM were virtually identical to those in AM. The AI-LM may represent AM that have been translocated from the alveolar space compartment to the conducting airways; it remains possible, however, that at least some of the AI-LM may have an airway origin. Regardless, the results of our phagocytic studies suggest AI-LM may functionally provide a protective phagocytic role in the conducting airways.

Biosynthesis and characterization of type X collagen in human fetal epiphyseal growth plate cartilage.

We examined in vitro collagen biosynthesis by organ cultures from human fetal epiphyseal growth plate cartilage. The biosynthetic products were characterized by NaCl fractional precipitation, limited proteolytic digestion, and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Organ cultures of human fetal epiphyseal growth plate cartilage synthesized large amounts of type X collagen in addition to type II, type IX, and type XI collagens. The individual polypeptide chains of human type X collagen migrated with an apparent M(r) of 45 kDa after proteolytic digestion with pepsin. The migration pattern of these molecules did not change when examined under reducing and nonreducing conditions, indicating that they did not contain intrahelical disfulfide bonds. Comparison of the rates at type X collagen biosynthesis at weeks 20 and 24 of human fetal development showed a marked increase of 24 weeks. Northern hybridization analysis of total RNA from freshly isolated epiphyseal growth plate chondrocytes with a cDNA corresponding to the carboxyl terminus of human type X collagen indicated that the developmental increase of type X collagen production is determined by pre-translational mechanisms.

Chronic depolarization prevents programmed death of sympathetic neurons in vitro but does not support growth: requirement for Ca2+ influx but not Trk activation.

Continuous exposure of many types of neurons in cell culture to elevated concentrations of K+ greatly enhances their survival. This effect has been reported to be mediated by a sustained rise of cytoplasmic free Ca2+ concentration caused by influx of Ca2+ through voltage-gated channels activated by K(+)-induced chronic depolarization. In this report we investigate the effects of elevated K+ on the programmed death that embryonic rat sympathetic neurons undergo in culture when deprived of NGF. Elevated K+ in the culture medium did not significantly prevent death of NGF-deprived cells until after the third day following plating of embryonic day 21 neurons. On the fifth day after plating, incrementally increasing K+ concentrations in the culture medium from 5 to 100 mM caused chronic depolarization of neurons and had a biphasic effect on survival of NGF-deprived cells. Enhanced survival was steeply related to membrane potential, increasing from no enhanced survival in cells held at potentials between -51 and -34 mV to 90-100% of control survival at about -21 mV. At potentials positive to -21 mV, survival decreased. Associated with the chronic depolarization was a sustained rise of steady-state free Ca2+ concentration that showed a biphasic relationship to membrane potential roughly similar to that exhibited by survival. Steady-state Ca2+ concentration increased with increasingly lower membrane potentials to a peak at about -23 mV (to approximately 240 nM from approximately 40 nM at about -51 mV) and then decreased at more positive potentials. The elevation of intracellular Ca2+ was largely blocked by dihydropyridine and phenylalkylamine Ca2+ channel antagonists and was potentiated by a dihydropyridine Ca2+ channel agonist. Neither the rise of Ca2+, or survival was affected by the Ca2+ channel antagonist, omega-conotoxin. Therefore, the Ca2+ elevation was probably caused by Ca2+ influx through L-type, but not N-type, channels. Antagonists of L channels blocked both survival and the sustained increase of steady-state free Ca2+ at similar concentrations, suggesting that the relevant factor determining survival of depolarized cells was Ca2+ influx rather than some other effect of depolarization. Surprisingly, however, there was no clear correlation between the sustained rise of Ca2+ and survival. Some membrane potentials that induced similar increases of Ca2+ concentration produced widely different levels of survival. While chronic depolarization promoted survival of neurons in the absence of NGF, cells supported in this manner showed little growth as measured by neurite extension, total cellular protein, and mean somal diameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Skeletal muscle-derived trophic factors prevent motoneurons from entering an active cell death program in vitro.

The purpose of the experiments reported here is to provide evidence that motoneurons (MTNs) isolated from chick embryo spinal cords go through an active process of cell death when deprived of trophic support in vitro. In order to analyze and characterize this process, MTNs were isolated with a metrizamide gradient technique and cultured in the presence of saturating concentrations of soluble muscle extract. When muscle extract was washed off from the cultures, MTNs entered a process of cell death that could be blocked with inhibitors of mRNA and protein synthesis. Two other additional criteria were used to define this process as an active one. First, ultrastructural analysis of MTNs dying as a consequence of muscle extract deprivation showed that some, but not all, of the MTNs displayed clear signs of apoptotic cell death. Those included cytoplasm condensation, fragmentation of chromatin, and preservation of cytoplasmic organelles. Second, internucleosomal degradation of DNA was detected in MTNs deprived of muscle extract. When DNA was analyzed by Southern hybridization techniques using digoxigenin-labeled genomic probes, a clear ladder pattern could be identified on muscle extract-deprived MTNs. The degradation of DNA upon trophic deprivation could be prevented by cycloheximide (CHX). In an attempt to characterize further the process of active cell death in MTNs, we found a time point of commitment to cell death of approximately 10 hr by using three different approaches: muscle extract deprivation plus readdition of muscle extract, muscle extract deprivation plus addition of CHX, and muscle extract deprivation plus addition of actinomycin D. Moreover, we show that MTNs deprived of trophic support from muscle extract but maintained alive with CHX could not be rescued from cell death by reading muscle extract if CHX was washed off the cultures within the first 15 hr of muscle extract deprivation. However, muscle extract alone was able to rescue MTNs that had been kept alive with CHX for periods of time longer than 24 hr after muscle extract deprivation. From these results we postulate that the activation of the cell death program after trophic deprivation is transient.

Transcriptional modulation of cartilage-specific collagen gene expression by interferon gamma and tumour necrosis factor alpha in cultured human chondrocytes.

To examine the possibility that cytokines produced in inflamed joint tissues may contribute to the loss of articular cartilage by causing inhibition of synthesis of cartilage-specific matrix macromolecules, we studied the effects of interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha), alone and in combination, on the expression of the genes for types-II, -IX and -XI collagens in cultured human chondrocytes. Chondrocytes isolated from human fetal epiphyseal cartilage by sequential enzymic digestions were cultured in the presence of IFN gamma (30 pM), TNF alpha (15 pM) or a combination of suboptimal concentrations of both cytokines (1.5 pM IFN gamma plus 0.3 pM TNF alpha). IFN gamma caused a maximal decrease of 23.3-32.6% in the biosynthesis of collagen by chondrocytes. TNF alpha was a more potent inhibitor causing a 42.8-45.3% decrease at one-half the concentration of IFN gamma. A synergistic inhibitory effect of 58.2% was observed with the combination of 1.5 pM IFN gamma plus 0.3 pM TNF alpha. Electrophoretic analysis of the biosynthesized proteins showed a co-ordinate decrease in the production of the three cartilage-specific collagen types II, IX and XI. These effects were accompanied by parallel changes in the steady-state levels of their corresponding mRNAs. In vitro transcription assays showed that the collagen inhibitory effects of the cytokines occurred largely at the transcriptional level. Similar effects of the cytokines were observed on biosynthesis of types-II, -IX and -XI collagens and steady-state mRNA levels for type-II collagen by chondrocytes obtained from adult articular cartilage. These observations indicate that IFN gamma and TNF alpha can induce a synergistic inhibition of the synthesis of cartilage-specific collagens by fetal and adult human chondrocytes and suggest that these effects may contribute to the articular cartilage loss that occurs in inflammatory joint diseases.

Effects of interferon-gamma and tumor necrosis factor alpha on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes.

OBJECTIVE: To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. METHODS: Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. RESULTS: The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. CONCLUSION: These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.

Calmodulin is involved in membrane depolarization-mediated survival of motoneurons by phosphatidylinositol-3 kinase- and MAPK-independent pathways.

In the present work, we find that the elevation of extracellular K+ concentration promotes the survival of chick spinal cord motoneurons in vitro deprived of any neurotrophic support. This treatment induces chronic depolarization of the neuronal plasma membrane, which activates L-type voltage-dependent Ca2+ channels, resulting in Ca2+ influx and elevation of the cytosolic free Ca2+ concentration. Pharmacological reduction of intracellular free Ca2+ or withdrawal of extracellular Ca2+ reversed the effects of depolarization on survival. The intracellular Ca2+ response to membrane depolarization developed as an initial peak followed by a sustained increase in intracellular Ca2+ concentration. The depolarizing treatment caused tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) without involving tyrosine kinase receptor activation. The calmodulin antagonist W13 inhibited the survival-promoting effect induced by membrane depolarization but not the tyrosine phosphorylation of MAPK. Moreover, depolarization did not induce phosphatidylinositol-3 kinase (PI-3K) phosphorylation in our cells, and the PI-3K inhibitor wortmannin did not suppress the survival-promoting effect of K+ treatment. These results suggest that calmodulin is involved in calcium-mediated survival of motoneurons through the activation of PI-3K- and MAPK-independent pathways.