Effects of pertussis toxin onα 1-agonist-mediated phosphatidylinositide turnover and myocardial cell hypertrophy in neonatal rat ventricular myocytes

Summary In neonatal rat ventricular myocytes pretreatment with pertussis toxin did not affect 1 M (–)-norepinephrine stimulation of inositol phosphates or myocardial cell hypertrophy as measured either by protein radiolabelling or by myocardial cell protein content. Thus guanine nucleotide protein (s) ADP-ribosylated by pertussis toxin do not play a role in two ₁-adrenoceptor-mediated processes, phosphatidylinositide turnover and induction of myocardial cell hypertrophy.     

Bacillus thuringiensis delta-endotoxin: evidence that toxin acts at the surface of susceptible cells

Enzymically activated delta-endotoxin of Bacillus thuringiensis covalently bound to Sephadex beads, has the same effect on insect cells in tissue culture as free toxin. The effect is prevented by antitoxin antibody and heat denaturation and is not due to a nonspecific protein effect, the beads, or toxin released from the beads. The toxin, therefore, probably acts at the cell surface.     

Sphingosine 1-phosphate antagonizes human neutrophil apoptosis via p38 mitogen-activated protein kinase

Sphingosine 1-phosphate (SPP) is associated with the regulation of apoptosis, although its role in neutrophil apoptosis remains poorly investigated. Here, we show that exogenous SPP antagonizes spontaneous and anti-Fas-induced apoptosis in neutrophils. Pre-treatment with pertussis toxin clearly reduced the apoptosis-inhibiting capacity of SPP. Consequently, we investigated the involvement of potential modulators of apoptosis that are activated downstream of Gi/G0-coupled receptors. Neither Akt activity nor change in basal activity of c-Jun N-terminal kinases was detected during apoptosis or after adding SPP. In contrast, there was a transient decrease in phosphorylation of both extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) during both spontaneous and anti-Fas-induced apoptosis. Although exogenous SPP reversed these reductions in kinase activity, experiments with inhibitors of ERK (PD98059) and p38 MAPK (SB203580) revealed that only SB203580 counteracted the effect of SPP. Thus, SPP counteracts neutrophil apoptosis via a Gi/G0 protein survival-signalling pathway that includes modulation of p38 MAPK activity.     

Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

Activation of CD47 receptors causes histamine secretion from mast cells

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcɛRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the βγ dimer of a G_i protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G_i protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G_i protein complex. Received 8 December 2008; received after revision 12 January 2009; accepted 29 January 2009     

Role of noncoding RNAs in regulation of cardiac cell death and cardiovascular diseases

Loss of functional cardiomyocytes is a major underlying mechanism for myocardial remodeling and heart diseases, due to the limited regenerative capacity of adult myocardium. Apoptosis, programmed necrosis, and autophagy contribute to loss of cardiac myocytes that control the balance of cardiac cell death and cell survival through multiple intricate signaling pathways. In recent years, non-coding RNAs (ncRNAs) have received much attention to uncover their roles in cell death of cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy, and heart failure. In addition, based on the view that mitochondrial morphology is linked to three types of cell death, ncRNAs are able to regulate mitochondrial fission/fusion of cardiomyocytes by targeting genes involved in cell death pathways. This review focuses on recent progress regarding the complex relationship between apoptosis/necrosis/autophagy and ncRNAs in the context of myocardial cell death in response to stress. This review also provides insight into the treatment for heart diseases that will guide novel therapies in the future.     

Bacillus thuringiensis δ-endotoxin: Evidence that toxin acts at the surface of susceptible cells

Summary Enzymically activated -endotoxin ofBacillus thuringiensis covalently bound to Sephadex beads, has the same effect on insect cells in tissue culture as free toxin. The effect is prevented by antitoxin antibody and heat denaturation and is not due to a nonspecific protein effect, the beads, or toxin released from the beads. The toxin, therefore, probably acts at the cell surface.     

Genetic and molecular approaches to synthesis and action of the yeast killer toxin

Summary The K1 killer toxin ofSaccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.     

Guanine nucleotides inhibit poly-L-arginine-induced membrane damage in polymorphonuclear leukocytes

Poly-L-arginine induces a strong release of the cytoplasmic enzyme lactate dehydrogenase from rabbit polymorphonuclear leukocytes, indicating that plasma membrane damage occurs. GTP gamma S, a stable guanine nucleotide, completely inhibits poly-L-arginine-induced LDH release whereas pretreatment of the cells with pertussis toxin gives a moderate inhibition. The results suggest that poly-L-arginine-induced plasma membrane damage is mediated by guanine nucleotide binding structures.     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998     

Selection for resistance toBacillus thuringiensis δ-endotoxin in an insect cell line (Choristoneura fumiferana)

Summary A cell line of the spruce budworm (FPMI-CF1) consists of a mixed population of cells that possess variable sensitivity to -endotoxin from crystals ofBacillus thuringiensis. A cell strain was selected from FPMI-CF1 which was resistant to the entomocidal protein extracted fromB. thuringiensis crystals. The resistant character was unstable, however, and could not be maintained in the absence of toxin during growth.The author wishes to thank S.S. Sohi (Forest Pest Management Institute, Canadian Forestry Service, Sault Ste. Marie, Ontario, Canada) for theC.fumiferana cell line, and L.I. Davidson (USDA) for his able technical assistance.     

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (K_V) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric β-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type K_V channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.     

Purification of a protein binding quinacrine and histrionicotoxin from membrane fragments rich in cholinergic receptors in Torpedo marmorata]

A protein is purified by differential centrifugation from membrane fragments rich in acetylcholine receptor prepared from Torpedo marmorata electric organ after dissolution by a mixture of non denaturing detergents. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and Coomassie blue staining the purified protein yields a single band of apparent molecular weight 43,000. Spectroscopic experiments carried out in the absence of Ca++ and detergents reveal that the 43 K protein interacts with the fluorescent local anesthetic quinacrine and with the frog toxin histrionicotoxin (apparent KD : 7 X 10(-7) M) but not with carbamylcholine and the alpha toxin from N. nigricollis.     

Minoxidil-induced cardiac hypertrophy in guinea pigs

To investigate whether during cardiac hypertrophy changes occur in contractile protein composition and in mechanical and energetic properties of the myocardium, contractile protein composition, isometric force and adenosine triphosphate (ATP) consumption were studied in control and hypertrophied guinea-pig hearts. Cardiac hypertrophy was induced by adding minoxidil (120 or 200 mg/l) to the drinking water. Protein analysis was performed by one-dimensional gel electrophoresis. The myosin heavy-chain (MHC) composition was determined in an enzyme-linked immunosorbent assay (ELISA). ATP consumption and force development were simultaneously measured during isometric contraction in chemically skinned trabeculae. Histochemical analysis of cross-sectional area of cardiomyocytes and interstitial space was performed on the left ventricular tissue of 200 mg/l minoxidil-treated and control guinea pigs. Minoxidil treatment (120 and 200 mg/l) significantly increased left ventricular dry weight normalized for body weight by 19 ± 4 and 24 ± 4%, respectively. No significant differences were found in the cellular cross-sectional area, while interstitial space was slightly decreased in minoxidil-treated hearts. In left ventricular trabeculae of 200 mg/l minoxidil-treated guinea pigs, ATPase activity was slightly less than in those of control guinea pigs, whereas force did not differ significantly. Calcium sensitivity of force and ATPase activity were not affected by minoxidil treatment. Gel electrophoresis revealed no difference in contractile protein composition, but a tendency towards a lower amount of α-MHC in the minoxidil-treated hearts was found in ELISA. Received 1 February 1999; accepted 15 March 1999     

The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC₈) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC₈ to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca²⁺ concentration or with an increase of intracellular Ca²⁺ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca²⁺-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A₂ (PLA₂) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low molar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA₂-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA₂ activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.     

What’s new in the renin-angiotensin system?

Activation of the type 1 angiotensin II receptor (AT(1)R) is associated with the aetiology of left ventricular hypertrophy, although the exact intracellular signalling mechanism(s) remain unclear. Transactivation of the epidermal growth factor receptor (EGFR) has emerged as a central mechanism by which the G protein-coupled AT(1)R, which lacks intrinsic tyrosine kinase activity, can stimulate the mitogen-activated protein kinase signalling pathways thought to mediate cardiac hypertrophy. Current studies support a model whereby AT(1)R-dependent transactivation of EGFRs on cardiomyocytes involves stimulation of membrane-bound metalloproteases, which in turn cleave EGFR ligands such as heparin-binding EGF from a plasma membrane-associated precursor. Numerous aspects of the 'triple membrane-passing signalling' paradigm of AT(1)R-induced EGFR transactivation remain to be characterised, including the identity of the specific metalloproteases involved, the intracellular mechanism for their activation and the exact EGFR subtypes required. Here we examine how 'hijacking' of the EGFR might explain the ability of the AT(1)R to elicit the temporally and qualitatively diverse responses characteristic of the hypertrophic phenotype, and discuss the ramifications of delineating these pathways for the development of new therapeutic strategies to combat cardiac hypertrophy.     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Enteric glial cells are susceptible to <Emphasis Type="Italic">Clostridium difficile</Emphasis> toxin B

Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1–10 ng/ml for 1.5–48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.