Modulation of Bradykinin-Induced Inositol Trisphosphate Release in a Novel Neuroblastoma X Dorsal Root Ganglion Sensory Neuron Cell Line (F-11)

In the mouse neuroblastoma x dorsal root ganglion hybrid cell line F-11, bradykinin receptor stimulation induced the release of inositol-1,4,5-trisphosphate (IP3) and inositol-1,4-bisphosphate (IP2). Maximal stimulation of [2-3H]IP3 and [2-3H]IP2 release by bradykinin in the absence of LiCl occurred at 7 (or less) and 15 s, respectively, with average levels of 5.7-(IP3) and 3.4-(IP2) fold of control values. The EC50 for bradykinin was 33 +/- 5 nM. IP3 and IP2 concentrations returned to basal levels approximately 1 min after bradykinin addition. Bradykinin-induced IP3 release was blocked by several novel bradykinin analogues. In particular, [D-Arg0]-Hyp3-Thi5,8-[D-Phe7]-bradykinin [Hyp, hydroxyproline; Thi, beta-(2-thienyl)-L-alanine] blocked IP3 production in a dose-dependent fashion. Several of these analogues alone showed little or no agonist activity. The bradykinin receptor may be coupled to phospholipase C via a GTP-sensitive protein (Gi or Go), as preincubation for 18-20 h with pertussis toxin decreased IP3 concentrations by 45%. Bradykinin is also known to modulate the concentrations of other second messengers in neurons, increasing the concentrations of Ca2+, diacylglycerol (DG), and cyclic GMP and decreasing the concentration of cyclic AMP. These second messengers modulated bradykinin-dependent IP3 release to varying degrees. A23187, a Ca2+ ionophore, produced a 37% decrease in IP3 concentration. 12-O-Tetradecanoylphorbol-13-acetate, which mimics the effects of DG and activates protein kinase C, inhibited IP3 release by 80%. Dibutyryl cyclic GMP produced little or no inhibition of IP3. [D-Ala2,D-Leu5]Enkephalin (DADLE), an opioid peptide that decreases cyclic AMP concentrations, likewise had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Trifluoperazine and chlorpromazine block secretion from human platelets evoked at basal cytoplasmic free calcium by activators of C-kinase

Trifluoperazine, chlorpromazine and other drugs to inhibit calmodulin-dependent processes are also known to inhibit protein kinase C. The effect of these agents on secretion evoked by known activators of C-kinase has been studied in human platelets loaded with the fluorescent Ca indicator, quin2 and preincubated with aspirin. The secretory response stimulated by phorbol ester and exogenous diacyglycerol, at basal levels of cytoplasmic free Ca²⁺, [Ca²⁺]_i, was suppressed by trifluoperazine, chlorpromazine and W-7, as was the secretion evoked by collagen that occurs without a change in [Ca²⁺]_i, The response to thrombin, which is accompanied by elevated [Ca²⁺]_i was barely affected. Modest elevation of [Ca²⁺]_i by Ca ionophore was able to overcome the inhibitory effect of these drugs on the response to phorbol ester, diacylglycerol, and collagen.     

Effects of toxicants on populations: A qualitative approach II. first order kinetics

System level effects exhibited by a population subjected to a chronic or an acute dose of toxicant are the emphasis of this study. A three dimensional model of a toxicant and a population, with state variables (the population biomass, the concentration of toxicant in an organism, and the concentration of toxicant in the environment) coupled by a linear dose-response function, is analyzed analytically. One of the main results presents sufficient conditions, in terms of a system level parameter, for the persistence, and for the extinction, of a population exposed to a chronic dose of toxicant. When depuration and degradation are negligible processes, the effects of toxicant accumulation associated with an acute exposure of a population are analyzed in some detail. Both persistence and extinction are shown to be viable behavior modes of a population in this biochemical setting.     

Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

Flagellum retraction and axoneme depolymerisation during the transformation of flagellates to amoebae inPhysarum

Summary The transformation ofPhysarum polycephalum flagellates to myxamoebae is characterised by disappearance of the flagellum. This transition, from the flagellate to the myxamoeba was observed by phase contrast light microscopy and recorded by time lapse video photography to determine whether flagellates shed their flagella or they are absorbed within the cell. In addition, the kinetics of flagellum disappearance were also studied. Our observations indicate that the flagellum was absorbed within the cell; the process occurred within seconds. Flagellum resorbtion was preceded by typical morphological cell changes. The shape of the nucleus altered and its mobility within the cell decreased. It was not possible to observe the flagellum within the cell with phase contrast video recordings. Thin section electron microscopy was used to study this intracellular phenomenon. Several stages of flagellum dissolution could be identified within the cell. The two most important stages were: an axoneme surrounded by the flagellar membrane within a plasma membrane lined pocket or vacuole and the naked axoneme without its membrane, free within the cell cytoplasm. The existence of cytoplasmic microtubules prevented identification of any further dissolution stages of the flagellum. A group of microtubules adjacent to the flagellum but within the cytoplasm was observed in flagellates and also in those cells which possesed enveloped axonemes. The flagellum did not dissociate from the kinetosomes before resorbtion.Immunofluorescence studies with the 6-11-B-1 monoclonal antibody indicated that acetylated microtubules exist in myxamoebae after transformation from flagellates for up to 40 min. Acetylated tubulin is not limited to the centrioles in these cells.