William Shain

Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

SummaryChanges in the fluorescence intensity of the dye 3-3′ dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10−9m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg2+ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.

Mechanisms of synaptic modulation.

Publisher Summary This chapter discusses the mechanism of synaptic modulation. Synaptic modulation is an observed change in the synaptic function. This change can occur by summation of a variety of physiological and biochemical cellular responses. Thus, the term, “synaptic modulation” does not make a precise statement and in this sense, is not analogous to the term, “synaptic transmission.” Synaptic transmission describes the transfer of a signal from one cell to another using mechanisms that are similar at all synapses. Synaptic transmission occurs at a particular location that can be described by morphologically pre- and postsynaptic specializations, which involves vesicular release of a neurotransmitter, and elicits a response from the postsynaptic cell through receptors that recognize the specific transmitter released. There are data inconsistent with this description, that is, release of amino acid transmitters that may not be vesicular, and adrenergic synapses that are difficult to describe morphologically. However, the term, “synaptic transmission” conveys both the description of the event as well as the mechanism involved.

Neurotransmitter modulation of prostaglandin E1-stimulated increases in cyclic AMP: II. Characterization of a cultured neuronal cell line treated with dibutyryl cyclic AMP

Abstract The ability of selected neurotransmitters to modulate PGE,-stimulated increases in cAMP was tested in the somatic cell hybrids TCX 17 and TCX 11 differentiated by growth in dibutyryl cAMP. PGE1 was shown to cause an increase in cellular cAMP. Carbachol, noradrenaline and dopamine inhibited the effect of PGE1, while 5-hydroxytryptamine had no effect. The carbachol inhibition is mediated by a muscarinic receptor since nicotinic antagonists failed to block carbachol while scopolamine reversed its effect. The noradrenaline inhibition was reversed by the antagonists phenoxy-benzamine and phentolamine, but not by the β-antagonists propranolol and dichloroisoproterenol. The dopamine inhibition was reversed by chlorpromazine and trifluoperazine. The dopamine agonist ET495 mimicked dopamine while apomorphine had little or no effect. These results obtained from differentiated cells are compared to those reported for exponential growth phase cells of the same cell line. Distinct differences were found with respect to the pharmacology of the noradrenaline and dopamine inhibition. Finally, the biochemical results are compared to the electrophysiological results reported for the cell lines. Neurotransmitter agents that modulate PGE, effects do not necessarily elicit membrane conductance changes, and, similarly, neurotransmitters that elicit an electrophysiological response do not inhibit PGE1-stimulated increases in cAMP. Dopamine elicits an electrophysiological response and inhibits the effects of PGE1. The possibility exists that a single receptor is mediating two cellular events.

Characterization of an 11,000-dalton beta-bungarotoxin: binding and enzyme activity on rat brain synaptosomal membranes.

The binding and phospholipase A2 activity of an 11,000-dalton beta-bungarotoxin, isolated from Bungarus multicincutus venom, have been characterized using rat brain subcellular fractions as substrates. 125I-labeled beta-bungarotoxin binds rapidly (k = 0.14 min-1 and 0.11 min-1), saturably (Vmax = 130.1 +/- 5.0 fmoles/mg and 128.2 +/- 7.1) fmoles/mg), and with high affinity (apparent Kd = 0.8 +/- 0.1 nM and 0.7 +/- 0.1 nM) to rat brain mitochondria and synaptosomal membranes, respectively, but not to myelin. The binding to synaptosomal membranes is inhibited by divalent cations and by pretreatment with trypsin. The binding results suggest that the toxin binds to specific protein receptor sites on presynpatic membranes. The 11,000-dalton toxin rapidly hydrolyzes synaptosomal membrane phospholipids to lysophosphatides and manifests relative substrate specificity in the order phosphatidyl ethanolamine greater than phosphatidyl choline greater than phosphatidyl serine. These results indicate that the 11,000-dalton beta-bungarotoxin is a phospholipase A2 and can use presynaptic membrane phospholipids as substrates. The binding, phospholipase activity and other biological properties of the 11,000-dalton toxin are contrasted with those of the beta-bungarotoxin found in highest concentration in the venom (the 22,000-dalton beta-bungarotoxin), and the two toxins are shown to have qualitatively similar properties. Finally the results are shown to support the hypothesis that beta-bungarotoxins act in a two-step fashion to inhibit transmitter release: first, by binding to a protein receptor site on the presynatic membrane associated with Ca2+ entry, and second, by perturbing through enzymatic hydrolyses the phospholipid matrix of the membrane and thereby causing an increase in passive Ca2+ permeability.

Evidence for a dopamine receptor antibody.

Summary Electrophysiological and biochemical studies have shown that the vertebrate neuronal somatic cell hybrid TCX11 expresses receptors for dopamine. Antiserum was produced in an effort to obtain an antibody to this dopamine receptor. Decomplemented antiserum reversibly blocked the electrophysiological dopamine response in TCX11. Two methods of purification yielded fractions which antagonized the response. The antiserum antagonized [ 3 H]-dopamine binding and the dopamine-sensitive adenylate cyclase in rat caudate homogenates. Dopamine responses were selectively blocked in the molluse Aplysia . These results suggest the presence of an antibody effective in altering dopamine receptor activity.

Neurotransmitter modulation of prosta-glandin E1-stimulated increases in cyclic AMP: I. Characterization of a cultured neuronal cell line in exponential growth phase

Abstract Two sympathetic ganglion cell X neuroblastoma somatic cell hybrids (TC × 11 and TC × 17) were found to have a PGE1-sensitive adenylyl cyclase which was inhibited in whole cells by carbachol, norepinephrine and dopamine. Serotonin and morphine were without effect, the latter despite the presence of opiate receptors. In the TC × 17 clone, carbachol produced a greater degree of inhibition (30 per cent of control levels) than norepinephrine or dopamine (55–65 per cent of control). The ed 50 for inhibition was of the order norepinephine > dopamine > carbachol (10−7, 3 × 10−7 and 10−6 M respectively). The inhibition by carbachol could be reversed by the muscarinic antagonists scopolamine and atropine, while the nicotinic antagonists α-bungarotoxin and d-tubocurarine were without effect. The inhibition by norepinephrine and dopamine possessed the following properties: (1) the inhibition was mimicked by phenylephrine but not by isoproterenol, nor by dopaminergic agonists apomorphine and ET495; (2) the α-antagonists phenoxybenzamine and phentolamine reversed the inhibition by norepinephrine and dopamine; and (3) chlorpromazine reversed the inhibition of cyclic AMPformation by dopamine. Other phenothiazines tested, trifluoperazine, and fluphenazine, had no effect. It is concluded that the TC × 17 clone expresses two classes of receptors capable of modulating PGE1. The cholinergic receptor is muscarinic and the catecholamine receptor has α-adrenergic properties.

Purification and biochemical characterization of an 11 000-dalton β-bungarotoxin

Abstract The chromatographic separation and biochemical characterization of a β-bungarotoxin is described. This toxin is isolated as the most basic eluting protein of Bungarus multicinctus venom when separated by column chromatography on CM-Sephadex C-25. The protein migrated as a single band on pH 4.3 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of this toxin was estimated to be 10 000 ± 1000 by analytical sedimentation analysis. This value was consistent with the electrophoretic mobility of the toxin in SDS-polyacrylamide gels. The amino acid composition of this 11 000-dalton β-bungarotoxin was similar to that of the 22 000-dalton β-bungarotoxin previously reported (Lee et al. (1972) J. Chromatogr. 72, 71–82; Kelly, R.B. and Brown, III, F.R. (1974) J. Neurobiol. 5, 135–150; Kondo et al. (1978) J. Biochem. Tokyo 83, 91–99), suggesting that the 11 000-dalton toxin may be one of the polypeptide chains of the large toxin. The 11 000-dalton β-bungarotoxin was toxic to mice when injected intravenously. Animals that received lethal doses exhibited hyperexcitability followed by ataxia, convulsions, and death. The minimum lethal dose was 0.12 μg/g body weight. This β-bungarotoxin exhibited Ca2+-dependent phospholipase A activity comparable to that of the 22 000-dalton β-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. The enzyme activity was destroyed by boiling for 3 min at pH 8.6. In addition, an enzymatically inactive quantity of the 11 000-dalton toxin, equivalent to five times the minimum lethal dose of enzymatically active toxin, was not lethal when injected into mice. To test whether phospholipase A activity is responsible for lethality, bee venom phospholipase A2 was injected into mice at similar and greater concentrations with no toxic effect. Thus, while phospholipase A activity may be required for the lethal effect of the 11 000-dalton β-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.

Sympathetic ganglion cell x neuroblastoma hybrids with opiate receptors

Abstract A number of cultured cells of nerve or smooth muscle origin have been surveyed for stereospecific binding of either [ 3 H] naloxone or [ 3 H]dihydromorphine. Of the cell lines tested, three somatic cell hybrids, TCX17, NX1 and Z5, exhibited substantial specific binding of both naloxone and dihydromorphine. In addition, specific binding of [ 3 H]naloxone by the hybrid clone 108cc15 was confirmed. The neuroblastoma, smooth muscle, glial, and other hybrid cell lines demonstrated little or no specific binding. Specific binding of [ 3 H] dihydromorphine is dose dependent. Dissociation constants for the three hybrid clones range from 15 to 90 nM and maximal binding values range from 75 to 225 fmoles/mg of protein. The order of effectiveness of other opiates in displacing bound ( 3 H]dihydromorphine in TCX17 cells agrees well with that found in brain. Sodium ions selectively inhibit agonist binding (dihydromorphine) but not antagonist binding (naloxone), while potassium and magnesium salts have little effect on either.