Paramedic rapid sequence intubation in patients with non-traumatic coma.

INTRODUCTION: Pre-hospital intubation by paramedics is widely used in comatose patients prior to transportation to hospital, but the optimal technique for intubation is uncertain. One approach is paramedic rapid sequence intubation (RSI), which may improve outcomes in adult patients with traumatic brain injury. However, many patients present to emergency medical services with coma of non-traumatic cause and the role of paramedic RSI in these patients remains uncertain. METHODS: The electronic Victorian Ambulance Clinical Information System was searched for the term 'suxamethonium' between 2008 and 2011. We reviewed the patient care records and included patients with suspected non-traumatic coma who were treated and transported by road-based paramedics. Demographics, intubation conditions, vital signs (before and after drug administration) and complications were recorded. Younger patients (<60 years) were compared with older patients. RESULTS: There were 1152 paramedic RSI attempts of which 551 were for non-traumatic coma. The success rate for intubation was 97.5%. There was a significant drop in blood pressure in younger patients (<60 years) with the mean systolic blood pressure decreasing by 16 mm Hg (95% CI 11 to 21). In older patients, the systolic blood pressure also decreased significantly by 20 mm Hg (95% CI 17 to 24). Four patients suffered brief cardiac arrest during pre-hospital care, all of whom were successfully resuscitated and transported to hospital. CONCLUSIONS: Paramedic RSI in patients with non-traumatic coma has a high procedural success rate. Further studies are required to determine whether this procedure improves outcomes.

Autotaxin (NPP-2), a metastasis-enhancing motogen, is an angiogenic factor.

Autotaxin [ATX (NPP-2)], originally isolated as a tumor motility-stimulating protein, has recently been shown to augment tumor aggressiveness. Specifically, atx-transfected, ras-transformed NIH3T3 cell lines have been shown to be more invasive, tumorigenic, and metastatic than mock-transfected ras-transformed control cells. In addition, the atx-transfected ras-transformed cell lines appeared to produce tumors that were much more hyperemic than those formed by appropriate control cells. This observation led to the present study, in which we demonstrate that ATX modulates angiogenesis both directly and indirectly. We have used a murine in vivo angiogenesis model in which treated Matrigel plugs are injected s.c. into athymic nude BALB/c mice. Using the same transfected cell lines as before, we found that mixing atx-transfected ras-transformed NIH3T3 cells into the Matrigel resulted in greater new blood vessel formation than control cells. Similarly, mixing purified ATX into the Matrigel resulted in new blood vessel formation within the plug, similar to that produced by vascular endothelial growth factor. Mechanistically, ATX is not a strong chemoattractant for human endothelial cells (HUVECs); however, it strongly stimulates motility in human coronary artery smooth muscle cells. In addition, ATX stimulates HUVECs grown on Matrigel to form tubules, much like vascular endothelial growth factor. Both of these normal cell types are shown to express and secrete ATX. In HUVECs, ATX expression is up-regulated by basic fibroblast growth factor in a time-dependent manner. This up-regulation also extends to secretion of enzymatically active protein, as demonstrated by Western blot analysis and quantification of type-1 phosphodiesterase activity. These results establish the presence of ATX in HUVECs and coronary artery smooth muscle cells and specify ATX as a novel angiogenic factor, suggesting that ATX could contribute to the metastatic cascade through multiple mechanisms, perhaps by supporting an invasive microenvironment for both normal and tumor cells.

Enzymatic activation of autotaxin by divalent cations without EF-hand loop region involvement.

Autotaxin (ATX) is a recently described member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins with potent tumor cell motility-stimulating activity. Like other NPPs, ATX is a glycoprotein with peptide sequences homologous to the catalytic site of bovine intestinal alkaline phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca(2+) or Mg(2+) was each demonstrated to increase the PDE activity of ATX in a concentration-dependent manner, whereas incubation of ATX with chelating agents abolished this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effect on V(max.) Three mutant proteins, Ala(740)-, Ala(742)-, and Ala(751)-ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to that of the wild-type protein. A deletion mutation of the entire loop region resulted in slightly reduced PDE activity but normal motility-stimulating activity. However, the PDE activity of this same deletion mutant remained sensitive to augmentation by cations, strongly implying that cations exert their effect by interactions outside of the EF-hand loop region.

A sensitive screening assay for secreted motility-stimulating factors.

Secreted motility-stimulating factors are often expressed and secreted at low concentrations that are difficult to detect by Northern or Western blot analysis. Autotaxin (ATX) is a tumor-secreted autocrine motility-stimulating factor that has been associated with tumor invasion and metastatic potential. Although ATX has a number of enzymatic activities, it is most sensitively detected by its induced chemotactic response. After transfecting ATX cDNA into NIH3T3 fibroblasts, we developed a motility-based method to screen the resulting cloned cells for secretion of active protein. We placed the cloned and transfected cells into the bottom wells of a modified Boyden chamber and placed responding cells (A2058 human melanoma cells) into the upper wells. After overnight incubation, the membrane that separated the two chambers was removed and stained. Simple densitometry measurements were sufficiently accurate to determine which clones secreted active protein. Utilizing this method, 4 positive cell lines were chosen out of 36 tested clones. Further tests on the expanded cell lines determined that all 4 were secreting ATX. Thus, this modified Boyden chamber assay appears to provide a rapid and highly adaptable means to identify cells that secrete motility-stimulating factors.

Cripto-1-induced increase in vimentin expression is associated with enhanced migration of human Caski cervical carcinoma cells.

Cripto-1 (CR-1), a member of the EGF-CFC peptide family, plays an essential role during mesoderm formation in vertebrates as well as in cancer development. Using cDNA gene expression array, Western blot, and indirect immunofluorescence, an increase in vimentin expression was demonstrated in CR-1-transfected human Caski cervical carcinoma cells compared to control vector-transfected cells. In parental Caski cells, recombinant CR-1 induced a dose-dependent increase of vimentin protein expression within 24 h. Since vimentin expression has been demonstrated to correlate with a more aggressive phenotype in human cervical cancer, the migration capacity of CR-1-transfected or CR-1-treated Caski cells was studied in the Boyden chamber assay. Compared to the vector-transfected or untreated Caski cells, CR-1-transfected cells or cells treated with recombinant CR-1 exhibit enhanced migration, both through collagen- and through gelatin-coated membranes. Additionally, CR-1 can function as a chemoattractant for Caski cells. These findings are of biological significance since CR-1 is overexpressed in several types of human carcinomas. The present data demonstrate that CR-1 can increase vimentin expression and modulate migration in human cervical carcinoma cells.

Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells.

Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression affects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two non-expressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The effect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little difference in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In MatrigelTM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This difference in invasiveness was not caused by differences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly amplified tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness.

Cyclocreatine inhibits stimulated motility in tumor cells possessing creatine kinase.

Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine), an analog of creatine and a substrate for creatine kinase (EC 2.7.3.2), inhibits the stimulated motility of tumor cells which possess creatine kinase. A2058-055 human melanoma cells, transfected with a creatine kinase gene, showed an 80-90% reduction in chemotactic response to type IV collagen when incubated overnight in the presence of 10 mM cyclocreatine (p < 0.0001 for n = 8 experiments). This inhibitory effect of cyclocreatine can be partially reversed by addition of creatine to the overnight cell treatment. Non-transfected cells, with very low levels of creatine kinase, were not significantly inhibited. Further experiments utilizing type IV collagen as attractant demonstrated that cyclocreatine inhibited the chemokinetic (91%) and the haptotactic (73%) responses and the in vitro invasion of A2058-055 cells through Matrigel-coated membranes (88%). In addition, motility stimulation of A2058-055 cells by either autotaxin or fibronectin was markedly inhibited by cyclocreatine. DU-145 prostatic tumor cells, which express endogenous creatine kinase, also have a reduced motility response to either autotaxin or epidermal growth factor induced motility in the presence of cyclocreatine.

Adenosine receptor mediates motility in human melanoma cells.

Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.

Nucleotide binding to autotaxin: crosslinking of bound substrate followed by lysC digestion identifies two labeled peptides.

Autotaxin (ATX) is a 125 kDa glycoprotein motility factor and exoenzyme which can catalyze the hydrolysis of either the alpha-beta or at the beta-gamma phosphodiester bond in ATP. Its motility stimulating activity requires an intact 5'-nucleotide phosphodiesterase (PDE) active site. Photolysis-dependent labeling of ATX with alpha-[32P]-8-N3-ATP, lysC digestion, and peptide HPLC resolved two radioactive fractions containing single peptides whose amino-terminal sequences were determined. Peptide A (T210FPNLYTLATG. . .) was derived from the PDE active site and peptide B (Y318GPFGPEMTNP. . .) was not previously known to be involved in any of the activities of ATX. The differential effect of NaCl concentration on the labeling of these two peptides, as well as on the two reaction types catalyzed by ATX, allows a classification of activities which predicts both the position of preferential peptide labeling by bound ATP and also the position of phosphodiester bond hydrolysis.

Autotaxin is an exoenzyme possessing 5'-nucleotide phosphodiesterase/ATP pyrophosphatase and ATPase activities.

Autotaxin (ATX) is an extracellular enzyme and an autocrine motility factor that stimulates pertussis toxin-sensitive chemotaxis in human melanoma cells at picomolar to nanomolar concentrations. This 125-kDa glycoprotein contains a peptide sequence identified as the catalytic site in type I alkaline phosphodiesterases (PDEs), and it possesses 5'-nucleotide PDE (EC 3.1.4.1) activity (Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Arestad, A., Cioce, V., Schiffmann, E., and Liotta, L. (1992) J. Biol. Chem. 267, 2524-2529; Murata, J., Lee, H. Y., Clair, T., Krutsch, H. C., Arestad, A. A., Sobel, M. E., Liotta, L. A., and Stracke, M. L. (1994) J. Biol. Chem. 269, 30479-30484). ATX binds ATP and is phosphorylated only on threonine. Thr210 at the PDE active site of ATX is required for phosphorylation, 5'-nucleotide PDE, and motility-stimulating activities (Lee, H. Y., Clair, T., Mulvaney, P. T., Woodhouse, E. C., Aznavoorian, S., Liotta, L. A., and Stracke, M. L. (1996) J. Biol. Chem. 271, 24408-24412). In this article we report that the phosphorylation of ATX is a transient event, being stable at 0 degrees C but unstable at 37 degrees C, and that ATX has adenosine-5'-triphosphatase (ATPase; EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Thus ATX catalyzes the hydrolysis of the phosphodiester bond on either side of the beta-phosphate of ATP. ATX also catalyzes the hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and the hydrolysis of NAD to AMP, and each of these substrates can serve as a phosphate donor in the phosphorylation of ATX. ATX possesses no detectable protein kinase activity toward histone, myelin basic protein, or casein. These results lead to the proposal that ATX is capable of at least two alternative reaction mechanisms, threonine (T-type) ATPase and 5'-nucleotide PDE/ATP pyrophosphatase, with a common site (Thr210) for the formation of covalently bound reaction intermediates threonine phosphate and threonine adenylate, respectively.

Autotaxin, tumor motility-stimulating exophosphodiesterase.

While nucleotides have a well-established role in intracellular metabolism, ATP and other nucleotides also have important extracellular roles in receptor-mediated signal transduction (34, 35). Extracellular or cell surface proteins capable of binding ATP and hydrolyzing phosphoester bonds of nucleotides are known to exist but their function has remained obscure. Our recent data point to a structure-function correlation between PDE activity and motility stimulation by ATX, indicating a biologically important functional role for the ecto/exophosdiesterases in the stimulation of cellular motility. Data from studies with PC-1 and gp130RB13-6 have suggested that cell surface PDE's may also play roles in cellular differentiation. Extracellular PDE activities, in combination with other nucleotidases, may result in ecto-nucleotidase cascades (36-38). These data suggest that ecto-/exo-enzymes may catalyze extracellular biochemical reactions that are important in the regulation of cell behavior.

Stimulation of tumor cell motility linked to phosphodiesterase catalytic site of autotaxin.

A family of extracellular type I phosphodiesterases has recently been isolated by cDNA cloning, but a physiological function linked to the phosphodiesterase active site has remained unknown. We now present evidence that the phosphodiesterase catalytic site, 201YMRPVYPTKTFPN213, is essential for the motility stimulating activity of autotaxin (ATX), one member of the exophosphodiesterase family. Native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds ATP noncovalently, and undergoes threonine phosphorylation. Homogeneously purified recombinant ATX, based on the teratocarcinoma sequence, retains these same activities. A single amino acid in the phosphodiesterase catalytic site, Thr210, is found to be necessary for motility stimulation, phosphodiesterase activity, and phosphorylation. Two mutant recombinant proteins, Ala210- and Asp210-ATX, lack motility stimulation and lack both enzymatic activities; Ser210-ATX possesses intermediate activities. Another mutation, with the adjacent lysine (Lys209) changed to Leu209-ATX, possesses normal motility stimulation with sustained phosphodiesterase activity but exhibits no detectable phosphorylation. This mutation eliminates the phosphorylation reaction and indicates that the dephosphorylated state is an active motility-stimulating form of the ATX molecule. By demonstrating that the phosphodiesterase enzymatic site is linked to motility stimulation, these data reveal a novel role for this family of exo/ecto-enzymes and open up the possibility of extracellular enzymatic cascades as a regulatory mechanism for cellular motility.

Cloning, chromosomal localization, and tissue expression of autotaxin from human teratocarcinoma cells.

Autotaxin, a potent human tumor cell motility-stimulating exophosphodiesterase, was isolated and cloned from the human teratocarcinoma cell line NTera2D1. The deduced amino acid sequence for the teratocarcinoma autotaxin has 94% identity to the melanoma-derived protein, 90% identity to rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), and 44% identity to the plasma cell membrane marker PC-I. Utilizing polymerase chain reaction screening of the CEPH YAC library, we localized the autotaxin gene to human chromosome 8q23-24. Northern blot analysis of relative mRNA from multiple human tissues revealed that autotaxin mRNA steady state expression is most abundant in brain, placenta, ovary, and small intestine.

Novel growth inhibitory effect of 8-Cl-cAMP is dependent on serum factors that modulate protein kinase A expression but not the hydrolysis of 8-Cl-cAMP.

8-Chloro-cyclic AMP (8-Cl-cAMP) exhibits growth inhibition in vitro and in vivo in a broad spectrum of cancer cell lines. We examined whether the hydrolyzed metabolite is involved in the effect of 8-Cl-cAMP. 8-Cl-cAMP (5 mu M, 3 days) exerted varying degrees (0-51%) of growth inhibition on the same cell line cultured in the medium containing different heat-inactivated serum. HPLC analysis neither detected 8-Cl-adenosine in the medium nor demonstrated any correlation between the decrease in 8-Cl-cAMP concentration in the medium and the degree of growth inhibition. The low K-m phosphodiesterase activity in the sera did not correlate with the varying degrees of growth inhibition. The cAMP-dependent protein kinase RI(alpha) subunit expression in the same cell line varied widely with the different serum supplements. 8-Cl-CAMP-, but not 8-Cl-adenosine-induced, growth inhibition correlated with the basal levels of RI(alpha) and specific downregulation of RI(alpha). 8-Cl-cAMP, but not 8-Cl-phenyl-thio-cAMP or N-6-benzyl-cAMP, inhibited cell growth in serum-free medium. These results show that 8-Cl-cAMP induces growth inhibition through down-regulation of protein kinase A type I in the absence of its hydrolysis.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

cDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals a homology with phosphodiesterases.

A human cDNA clone encoding autotaxin, a tumor cell motility-stimulating protein, reveals that this protein is an ecto/exo-enzyme with significant homology to the plasma cell membrane differentiation antigen PC-1. ATX is a 125-kDa glycoprotein, previously isolated from a human melanoma cell line (A2058), which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations. Affinity-purified antipeptide antibodies to the ATX peptide, ATX-102, were employed to screen an A2058 cDNA expression library made in lambda gt11. The partial cDNA sequence which was obtained was then extended by utilizing reverse transcriptase on total cellular RNA followed by polymerase chain reaction amplification. The isolated cDNA clone contained 3251 base pairs, and the mRNA message size was approximately 3.3 kilobases. The deduced amino acid sequence of autotaxin matched 30 previously sequenced peptides and comprised a protein of 915 amino acids. Data base analysis of the ATX sequence revealed a 45% amino acid identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I phosphodiesterase expressed on the surface of activated B cells and plasma cells. ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate p-nitrophenyl thymidine-5'-monophosphate. Autotaxin now defines a novel motility-regulating function for this class of ecto/exo-enzymes.

The RI alpha subunit of protein kinase A controls serum dependency and entry into cell cycle of human mammary epithelial cells.

MCF-10A, a nontransformed mammary epithelial cell line, requires a mixture of hormones and growth factors for optimal cell proliferation. In this report we show that when MCF-10A cells are cultured in serum free medium they become quiescent and accumulate in G0/G1 phases of the cell cycle. Following addition of complete medium to quiescent cells, MCF-10A cells enter into the S phase within 15-18 h and resume the cell cycle distribution of proliferating cells within 24 h. Measurement of RI alpha subunit of the cAMP-dependent protein kinase (PKA) shows a 10- to 15-fold increase in protein levels at 6 h following complete medium addition, thus preceding cell entry into the S phase. Retroviral vector-mediated overexpression of RI alpha, but not of RII beta or C alpha subunits of PKA, enables MCF-10A cells to grow in serum-free medium. In addition, RI alpha downregulation by specific antisense oligodeoxynucleotide treatment or following infection with a retroviral vector containing the RI alpha cDNA in antisense orientation determines growth arrest of proliferating MCF-10A cells and is able to partially block S phase entry of quiescent MCF-10A cells following complete medium addition. These results suggest that RI alpha/PKAI is involved in the control of cell cycle progression of mammary epithelial cells at a G1 to S transition border, and that its overexpression is able to overcome serum and growth factors requirement for cell proliferation.

Retroviral vector-mediated overexpression of the RII beta subunit of the cAMP-dependent protein kinase induces differentiation in human leukemia cells and reverts the transformed phenotype of mouse fibroblasts.

We have recently shown, using antisense strategy, that the RII beta regulatory subunit of cAMP-dependent protein kinase is essential for cAMP-induced growth inhibition and differentiation of HL-60 human leukemia cells. We constructed a retroviral vector for RII beta (MT-RII beta) by inserting human RII beta complementary DNA into the OT1521 retroviral vector plasmid that contains an internal mouse metallothionein-1 promoter and a neomycin resistance gene. The PA317 packaging cell line was then transfected with MT-RII beta plasmid to produce the amphotrophic stock of MT-RII beta retroviral vector. The infection with MT-RII beta and treatment with CdCl2 brought about growth arrest in HL-60 human leukemia and Ki-ras-transformed NIH 3T3 clone DT cells in monolayer culture with no sign of toxicity. The growth inhibition correlated with the expression of RII beta and accompanied changes in cell morphology; cells became flat, exhibiting enlarged cytoplasm. The growth of these cells in semisolid medium (anchorage-independent growth) was almost completely suppressed. In contrast, overexpression of the RI alpha subunit of protein kinase enhanced the cell proliferation in DT cells. The MT-RII beta-infected cells exhibited an increased sensitivity toward treatment with cAMP analogues, such as 8-Cl-cAMP and N6-benzyl-cAMP, as compared with the parental noninfected cells. In MT-RII beta HL-60 cells, N6-benzyl-cAMP treatment greatly enhanced the expression of monocytic surface markers. These results suggest that the RII beta cAMP receptor, by binding to its ligand, cAMP, acts as a tumor suppressor protein exerting growth inhibition, differentiation, and reverse transformation.