Comparison of the Effects of Endotracheal Intubation of Wistar Rats Using the Conventional Technique vs. a New Modified Technique Using a 3D Mouth-Piece.

AIMS: Endotracheal intubation in rats is challenging due to the difficult anatomical characteristics of the airway. The success rate at first attempt is low and airway damage is a common complication. We aimed to compare and evaluate the conventional intubation method with a modified procedure using an inclined plate, headlamp (700-Lumen), and 3D mouth-piece designed with a 20° curvature. Both techniques were conducted by laboratory personnel with and without previous experience in airway management of laboratory rats. MATERIAL AND METHODS: In this study, we used 36 Wistar rats of both genders. Three groups of laboratory personnel (anesthesiologists, medical students, and laboratory technicians) performed both endotracheal intubation techniques, i.e., blind intubation at supine position and endotracheal intubation at 70° supine position with a 3D mouth-piece and direct illumination of the glottis. RESULTS: The modified technique had a significantly higher success rate and shorter procedure duration. Moreover, there was no significant difference in the procedure duration between personnel with and without previous training in airway management. CONCLUSION: Previous knowledge and experience in airway management are required when performing conventional endotracheal intubation; moreover, its success rate is low. Contrastingly, using proper instruments and the 3D mouth-piece facilitated easier and quicker airway management regardless of previous experience.

16K prolactin induces NF-kappaB activation in pulmonary fibroblasts.

The amino-terminal 16 kDa fragment of prolactin (16K PRL) promotes the expression of the inducible isoform of nitric oxide synthase (iNOS) accompanied by the production of nitric oxide (NO) by rat pulmonary fibroblasts. The present study was designed to elucidate whether the mechanism by which 16K PRL promotes iNOS expression involves the activation of nuclear factor-kappa B (NF-kappaB), a key transcription factor for iNOS induction. 16K PRL stimulated DNA-binding activity of NF-kappaB in pulmonary fibroblasts as demonstrated by gel shift assays. Likewise, fluorescence immunocytochemistry showed that 16K PRL promotes nuclear translocation of the p65 subunit of NF-kappaB. Finally, treatment with 16K PRL induced the degradation of the NF-kappaB inhibitor kappaB-beta (IkappaB-beta), and such degradation was prevented by blocking IkappaB-beta phosphorylation. Altogether, these results show that 16K PRL activates NF-kappaB nuclear translocation via the phosphorylation and degradation of IkappaB-beta. These findings are consistent with NF-kappaB being part of the signal transduction pathway activated by 16K PRL to induce iNOS expression.

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-kappaB and MAPK activation through separate signaling pathways.

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, beta1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via beta1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor kappaB (NF-kappaB) activation was then analyzed. We found that integrins activated both NF-kappaB and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-kappaB activation. In contrast, a dominant negative mutant of Rac completely prevented NF-kappaB activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-kappaB is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Rho augmented NF-kappaB activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-kappaB, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

Ceramide mimics tumour necrosis factor-alpha in the induction of cell cycle arrest in endothelial cells. Induction of the tumour suppressor p53 with decrease in retinoblastoma/protein levels.

Tumour necrosis factor (TNF)-alpha induces a transient increase in N-octanoylsphingosine (C8-ceramide) which has been postulated as an intracellular mediator in TNF-alpha signalling. We tested the ability of C8-ceramide to reproduce the TNF-alpha-mediated interference with endothelial cell proliferation and DNA synthesis. TNF-alpha (10 ng.mL-1) and C8-ceramide (20 microM) inhibited the incorporation of [3H]thymidine into DNA and led to an accumulation of cells in the G1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF-alpha and C8-ceramide induced increased expression of p53. Treatment with TNF-alpha or C8-ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF-alpha prevented RB phosphorylation whereas C8-ceramide did not interfere with this process, suggesting that it follows a ceramide-independent pathway.

Pancreatic beta cells synthesize and secrete nerve growth factor.

Differentiation and function of pancreatic beta cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic beta cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in beta cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic beta-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

Interference with c-myc expression and RB phosphorylation during TNF-mediated growth arrest in human endothelial cells.

Incorporation of [3H]-thymidine into DNA in non-synchronized cultures of human endothelial cells was blocked by a 24 h exposure to TNF in a dose dependent manner that resulted in accumulation of cells in G1, as assayed by flow cytometry analysis of DNA content. Proliferation restarted when cells were replated in the absence of TNF. Northern analysis of c-myc mRNA in synchronized untreated cultures showed a transient increase previous to DNA synthesis that was decreased with TNF treatment. Western analysis of the retinoblastoma gene product RB in untreated synchronized cultures showed reduced electrophoretic mobility during the transition from G1 to S, congruent with RB inactivation by phosphorylation. TNF treatment prevented RB retardation and reduced total levels of RB protein. Taken together our results show that the TNF-mediated block of endothelial proliferation correlates with deficient activation of the G1 events necessary for entry into S, despite the presence of serum and endothelial mitogens.

Ceramide reproduces late appearance of oxidative stress during TNF-mediated cell death in L929 cells.

Tumor necrosis factor alpha mediated cell death in L929 cells correlates with a late increase in reduction of the superoxide scavenger 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), suggesting an increase in MTT reduction per viable cell. This effect was studied in two TNF-sensitive and in five different TNF-resistant clones. Within 36 hrs TNF promoted a 7-fold increase in the reduction of MTT in TNF-sensitive cells. Exogenous ceramide induced a similar effect prior to cell death. Four of the five TNF-resistant clones were also resistant to ceramide and displayed no increase in MTT reduction with either TNF or ceramide. The remaining TNF-resistant clone was sensitive to ceramide, displaying an increase in MTT reduction. Our results suggest a late increase in superoxide production prior to cellular destruction during TNF and ceramide mediated cell death and support the notion that ceramide can serve as a second messenger for TNF in cell death.

Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the receptor complex.

T beta R-II (transforming growth factor beta [TGF-beta] type II receptor) is a transmembrane serine/threonine kinase that acts as the primary TGF-beta receptor. Ligand binding to T beta R-II leads to the recruitment and phosphorylation of T beta R-I, a distantly related transmembrane kinase that acts as a downstream signaling component. T beta R-I phosphorylation by T beta R-II is shown here to be essential for signaling. A mutant T beta R-II that binds ligand but lacks signaling activity was identified. This mutant was identified by screening with a TGF-beta-inducible vector a series of mink lung epithelial cell clones that have normal TGF-beta binding activity but have lost antiproliferative and transcriptional responses to TGF-beta. When transiently cotransfected with T beta R-II, one of these cell lines, S-21, recovered TGF-beta responsiveness. cDNA cloning and sequencing of T beta R-II from S-21 cells revealed a point mutation that changes proline 525 to leucine in kinase subdomain XI. A recombinant receptor containing this mutation, T beta R-II(P525L), is similar to wild-type T beta R-II in its abilities to bind ligand, support ligand binding to T beta R-I, and form a complex with T beta R-I in vivo. T beta R-II(P525L) has autophosphorylating activity in vitro and in vivo; however, unlike the wild-type receptor, it fails to phosphorylate an associated T beta R-I. These results suggest that T beta R-II(P525L) is a catalytically active receptor that cannot recognize T beta R-I as a substrate. The close link between T beta R-I transphosphorylation and signaling activity argues that transphosphorylation is essential for signal propagation via T beta R-I.

Cachectin/TNF-mediated lactate production in cultured myocytes is linked to activation of a futile substrate cycle.

The cytokine cachectin/TNF induces a rapid increase in lactate production and in glucose metabolism in L6 myocytes in culture; glucose uptake was maximal after 17 h, while elevated glucose utilization and lactate production persisted for up to 32 h. These increases are suggestive of increased glycolytic activity, and were associated with a 10% decrease in cellular oxygen consumption and a comparable decrease in the production of 14C-labelled CO2 from 14C-labelled glucose. This decrease in aerobic metabolism, however, could account for only a small fraction of the energetic requirement for increased glycolytic activity. Furthermore, maximal stimulation of pyruvate dehydrogenase (PDH) by dichloroacetate (DCA) treatment in conjunction with cachectin/TNF abolished lactate production, but increased glucose uptake persisted. Taken together, this suggests that the primary effect of cachectin/TNF on myocyte carbohydrate metabolism is to increase glycolysis. Correspondingly, we postulated that cachectin/TNF must activate one or more ATP-depleting cellular processes to account for the lack of feed-back inhibition on glycolysis by the ATP produced. This led to the identification of a futile substrate cycle between fructose 6-phosphate and fructose 1,6-bisphosphate as a novel energy sink that is activated by cachectin/TNF. Cachectin/TNF treatment led to increased activity of both phosphofructokinase (PFK) and fructose bisphosphate phosphatase (FBP) in myocytes in culture, detectable after 1 h of incubation and persisting for up to 16 h. The possible role of cachectin/TNF-mediated futile substrate cycling in increased glycolytic activity, increased energy expenditure, heat production and tissue wasting during bacterial infections is discussed.

TGF beta signals through a heteromeric protein kinase receptor complex.

Transforming growth factor beta (TGF beta) binds with high affinity to the type II receptor, a transmembrane protein with a cytoplasmic serine/threonine kinase domain. We show that the type II receptor requires both its kinase activity and association with another TGF beta-binding protein, the type I receptor, to signal growth inhibition and early gene responses. Receptors I and II associate as interdependent components of a heteromeric complex: receptor I requires receptor II to bind TGF beta, and receptor II requires receptor I to signal. This mode of operation points to fundamental differences between this receptor and the protein-tyrosine kinase cytokine receptors.

Transforming growth factor beta induces myoblast differentiation in the presence of mitogens.

Transforming growth factor beta 1 (TGF-beta 1) added to L6E9 rat skeletal myoblasts in mitogen-rich medium induces a rapid decrease in c-myc expression and delays progression through the G1 phase of the cell cycle. This growth inhibitory response is followed by cell commitment to terminal differentiation with elevated expression of myogenin muscle determination genes, induction of muscle-specific proteins, and formation of multinucleated myotubes. These results suggest that TGF-beta 1 may act as a physiological inducer of myogenic differentiation in mitogen-rich environments, and its otherwise reversible growth inhibitory effect may become permanent if coupled to induction of terminal differentiation.

Transforming growth factor-beta.

This chapter has described some of the most salient features of the biology of the TGF-beta s. The TGF-beta s are of great interest as growth inhibitors, regulators of cell phenotype and regulators of cell adhesion. The various TGF-beta isoforms are highly conserved and display a complex pattern of interactions with multiple membrane receptor components. Activation of these receptors leads to inhibition of epithelial cell proliferation by a mechanism that may involve proteins related to the growth suppressor, RB. TGF-beta receptors are also coupled to mechanisms that control expression of differentiation commitment genes and differentiated cell functions. TGF-beta can affect cell proliferation and differentiation through indirect mechanisms involving regulation of expression of cytokines, extracellular matrix molecules and their respective receptors. These responses strongly influence the growth and phenotype of an array of cell types. Excess or reduced TGF-beta activity may contribute to the pathogenesis of certain fibrotic disorders and certain hyperproliferative disorders including cancer, respectively.

Early gene responses to transforming growth factor-beta in cells lacking growth-suppressive RB function.

The growth-suppressive function of the retinoblastoma susceptibility gene product, RB, has been implicated in the mediation of growth inhibition and negative regulation of certain proliferation related genes by transforming growth factor-beta 1 (TGF-beta 1). Early gene responses to TGF-beta 1 were examined in order to determine their dependence on the cell cycle and on the growth-suppressive function of RB. TGF-beta 1, which rapidly elevates the steady-state level of junB and PAI-1 mRNAs and decreases that of c-myc mRNA, induces these responses in S-phase populations of Mv1Lu lung epithelial cells containing RB in a phosphorylated state. Since in this state RB is presumed to lack growth-suppressive activity, the response to TGF-beta 1 was also examined in DU145 human prostate carcinoma cells whose mutant RB product lacks growth-suppressive function. In these cells, TGF-beta 1 also decreases c-myc expression at the transcription initiation level. These results suggests that the c-myc, junB, and PAI-1 responses to TGF-beta 1 are not restricted to the G1 phase of the cell cycle and that down-regulation of c-myc expression by TGF-beta 1 can occur through a mechanism independent from the growth-suppressive function of RB.

The role of cachectin/TNF and other cytokines in sepsis.

A traditional view has been that bacterial products, such as endotoxins from gram negative bacteria, have a direct deleterious effect on the host, resulting in fever, hypermetabolism, anorexia, and tissue damage. In recent years, however, it has been shown that endogenous products of the host, secreted by macrophages and other cellular elements of the immune system, act as mediators in activating the metabolic and other physiological changes characteristic of the sepsis syndrome. We will review in depth various aspects of the major, central mediator, i.e., tissue necrosis factor (TNF)/cachectin, and also briefly discuss the interleukins IL-6 and IL-1.

Characterization of high molecular weight glycosylated forms of murine tumor necrosis factor.

Tumor necrosis factor (TNF) is synthesized as a prohormone with an unusually long and atypical signal sequence which is absent from the mature secreted cytokine. In addition to mature 17 kDa TNF, LPS-stimulated murine macrophages secrete at least seven TNF-like proteins (isoforms) of differing electrophoretic mobility which appear as a "ladder" on SDS-PAGE. We here present data indicating that these isoforms derive not from sequential clipping of propiece fragments, but rather from differential glycosylation at sites on the mature hormone. Selected isoforms have been isolated and purified by sequential chromatographic and electrophoretic steps. NH2-terminal sequence analysis of two of these isoforms reveal sequences identical to that of mature 17 kDa murine TNF. Characterization of the secretory products of tunicamycin-treated. LPS-stimulated murine macrophages indicate that the "ladder" complex reflects differential glycosylation of mature 17 kDa TNF. Digestion of purified isoforms with a battery of glycosidic enzymes indicate that secreted forms of murine TNF contain both sialic acid and asparagine(N)-linked chains. The biological significance of this heterogeneity is not known.

Effect of adenosine and inosine on ureagenesis in hepatocytes.

Adenosine and inosine produced a dose-dependent stimulation of ureagenesis in isolated rat hepatocytes. Hypoxanthine, xanthine and uric acid were without effect. Half-maximally effective concentrations were 0.08 microM for adenosine and 5 microM for inosine. Activation of ureagenesis by both nucleosides had the following characteristics: (a) it was observed with either glutamine or (NH4)2CO3, provided that glucose was present; (b) it was not detected when glucose was replaced by lactate plus oleate; (c) it was mutually antagonized by glucagon, but not by adrenaline; and (d) it was dependent on Ca2+. We suggest that the action of adenosine and inosine on ureagenesis might be of physiological significance.

Effect of endotoxin-induced monokines on glucose metabolism in the muscle cell line L6.

Exposure of fully differentiated L6 myotubes to a crude monokine preparation from endotoxin-stimulated RAW 264.7 cells resulted in a rapid and substantial (70%) increase in fructose 2,6-bisphosphate concentration coincident with a depletion of cellular glycogen and an increased lactate production. During the time required for glycogen depletion (3 hr), stimulation of 3-O-methyl-D-glucose and 2-deoxy-D-glucose uptake was initiated and observed to reach a maximum enhancement of 200% 12-15 hr later. The monokine had no effect on the Km value for 2-deoxy-D-glucose uptake (1.1 mM), while Vmax was increased from 912 to 2400 pmol/min per mg of protein. The increase was cytochalasin B inhibitable and was dependent on protein synthesis. Photoaffinity labeling and equilibrium binding studies with [3H]cytochalasin B support the hypothesis that this increase in hexose transport was due to an increase in hexose transporters present in the plasma membrane. Purified recombinant interleukin-1 alpha had no effect on hexose transport, whereas purified recombinant cachetin/tumor necrosis factor did stimulate hexose uptake, with half-maximal stimulation occurring at 36 nM. Although cachetin accounts for most of the biological activity associated with the crude monokine preparations, it is not the only monokine capable of inducing glucose transport in L6 cells. Specific immunoabsorption of cachectin/tumor necrosis factor from the crude monokine preparation revealed a monokine that had a similar bioactivity at extremely low concentrations on L6 cells.

Shock and tissue injury induced by recombinant human cachectin.

Cachectin (tumor necrosis factor), a protein produced in large quantities by endotoxin-activated macrophages, has been implicated as an important mediator of the lethal effect of endotoxin. Recombinant human cachectin was infused into rats in an effort to determine whether cachectin, by itself, can elicit the derangements of host physiology caused by administration of endotoxin. When administered in quantities similar to those produced endogenously in response to endotoxin, cachectin causes hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours, as a result of respiratory arrest. Hyperglycemia and hyperkalemia were also observed after infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent on gross and histopathologic examination, along with ischemic and hemorrhagic lesions of the gastrointestinal tract, and acute renal tubular necrosis. Thus, it appears that a single protein mediator (cachectin) is capable of inducing many of the deleterious effects of endotoxin.