UMLS:C0016632 - FACTA Search

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Query: UMLS:C0016632 (Fox)
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Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90 000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.
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PMID:Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport. 12 Feb

The detergent-insoluble cytoskeleton of the resting human blood platelet contains approximately 2,000 actin filaments approximately 1 micron in length crosslinked at high angles by actin-binding protein and which bind to a spectrin-rich submembrane lamina (Fox, J., J. Boyles, M. Berndt, P. Steffen, and L. Anderson. 1988. J. Cell Biol. 106:1525-1538; Hartwig, J., and M. DeSisto. 1991. J. Cell Biol. 112:407-425). Activation of the platelets by contact with glass results within 30 s in a doubling of the polymerized actin content of the cytoskeleton and the appearance of two distinct new actin structures: bundles of long filaments within filopodia that end at the filopodial tips (filopodial bundles) and a circumferential zone of orthogonally arrayed short filaments within lamellipodia (lamellipodial network). Neither of these structures appears in cells exposed to glass with cytochalasin B present; instead the cytoskeletons have numerous 0.1-0.3-microns-long actin filament fragments attached to the membrane lamina. With the same time course as the glass-induced morphological changes, cytochalasin-sensitive actin nucleating activity, initially low in cytoskeletons of resting platelets, increases 10-fold in cytoskeletons of thrombin-activated platelets. This activity decays with a time course consistent with depolymerization of 0.1-0.3-microns-long actin filaments, and phalloidin inhibits this decay. Cytochalasin-insensitive and calcium-dependent nucleation activity also increases markedly in platelet extracts after thrombin activation of the cells. Prevention of the rise in cytosolic Ca2+ normally associated with platelet activation with the permeant Ca2+ chelator, Quin-2, inhibits formation of lamellipodial networks but not filopodial bundles after glass contact and reduces the cytochalasin B-sensitive nucleation activity by 60% after thrombin treatment. The filopodial bundles, however, are abnormal in that they do not end at the filopodial tips but form loops and return to the cell body. Addition of calcium to chelated cells restores lamellipodial networks, and calcium plus A23187 results in cytoskeletons with highly fragmented actin filaments within seconds. Immunogold labeling with antibodies against gelsolin reveals gelsolin molecules at the ends of filaments attached to the submembrane lamina of resting cytoskeletons and at the ends of some filaments in the lamellipodial networks and filopodial bundles of activated cytoskeletons. Addition of monomeric actin to myosin subfragment 1-labeled activated cytoskeletons leads to new (undecorated) filament growth off the ends of filaments in the filopodial bundles and the lamellipodial network. The simplest explanation for these findings is that gelsolin caps the barbed ends of the filaments in the resting platelet. Uncapping some of these filaments after activation leads to filopodial bundles.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanisms of actin rearrangements mediating platelet activation. 132 75

omega-Conotoxin GVIA (omega-CgTx-VIA) is a 27 amino acid peptide from the venom of the fish-hunting snail, Conus geographus, that blocks voltage-activated Ca channels. The characterization of a biologically active, homogeneous 125I-labeled monoiodinated Tyr22 derivative of omega-conotoxin GVIA and its use in binding and cross-linking studies are described. The 125I-labeled toxin is specifically cross-linked to a receptor protein with an apparent Mr of 135,000. The stoichiometry between omega-conotoxin and nitrendipine binding sites in different chick tissues was determined. Skeletal muscle has a high concentration of [3H]nitrendipine binding sites (greater than 1000 fmol/mg) but no detectable omega-conotoxin sites (less than 7 fmol/mg). Brain microsomes have both binding sites, but omega-conotoxin targets are in excess. These results, combined with recent electrophysiological studies (E. W. McCleskey, A. P. Fox, D. Feldman, L. J. Cruz, B. M. Olivera, R. W. Tsien, and D. Yoshikami, unpublished results), define four types of Ca channels in chick tissues, N, T, Ln (omega sensitive), and Lm (omega insensitive), and are consistent with the hypothesis that the alpha-subunits of certain neuronal Ca2+ channels (Ln, N) are the molecular targets of omega-conotoxin GVIA.
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PMID:Characterization of the omega-conotoxin target. Evidence for tissue-specific heterogeneity in calcium channel types. 243 55

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca2+]i. Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440-442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[gamma-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[beta-thio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]i at rest but caused a partial blunting of the initial [Ca2+]i peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.
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PMID:Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons. 243 97

1. T-, and L-type Ca2+ channels were studied in cell-attached patch recordings from the cell bodies of chick dorsal root ganglion neurones. All experiments were performed with isotonic BaCl2 (110 mM) in the recording pipette and with isotonic potassium aspartate in the bathing solution to zero the cell membrane potential. 2. L-type channels are distinguished by a unitary slope conductance of 25 pS, activation over the range of membrane potentials between 0 and +40 mV, little inactivation over the course of a 136 ms depolarization, and availability for opening even at depolarized holding potentials (h.p. greater than -40 mV). L channels show a predominant mode of gating (mode 1) characterized by brief openings (approximately 1 ms), occasionally interspersed with another pattern of gating characterized by much longer openings (mode 2). 3. The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 promotes mode 2 activity and shifts the voltage dependence of L-type channel activation towards more negative potentials. It leaves the unitary current-voltage relation unchanged. 4. Nifedipine, a DHP Ca2+ antagonist, strongly inhibits L-type channel activity through an increase in the proportion of blank sweeps. 5. T-type Ca2+ channels are distinguished by a much smaller unitary slope conductance (8 pS) and by activation and inactivation over relatively negative ranges of potential. Inactivation is complete by the end of 136 ms pulses to test potentials beyond -20 mV. 6. N-type Ca2+ channels are distinguished by an intermediate unitary slope conductance (13 pS), and by activation over a range of potentials between those of T- and L-type channels. Inactivation of N-type channels takes place over an exceptionally broad range of holding potentials (-80 to -20 mV). 7. Cell-attached patch data on the voltage dependence of activation and inactivation of T- and N-type channels are in excellent agreement with results from whole-cell recordings (Fox, Nowycky & Tsien, 1987) if allowances are made for variations in external surface potential. 8. Patches containing one or two channels of a single type were used for analysis of gating kinetics. The predominant pattern of activity for each of the channel types is an exponential distribution of relatively brief (approximately 1 ms) openings, and a bi-exponential distribution of short and long closings. 9. Patches containing all possible combinations of channel types were observed. However, preliminary evidence suggests that channels are distributed unevenly over the cell body; clustering of N-type channels is particularly prominent.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Single-channel recordings of three types of calcium channels in chick sensory neurones. 245 Oct 17

1. Ca2+ channels were studied in cultured glomerulosa cells from the rat adrenal gland. The whole-cell configuration of the patch-clamp technique was used. Cs+-filled pipettes were used in order to block K+ channels. 2. Three Ca2+ components were found, namely, T, L and N, according to the nomenclature proposed by Nowycky, Fox & Tsien (1985). The T-component was a fast transient component activated in the range -60 to -40 mV; the L-component did not inactivate for a sustained depolarization and activated at voltages around -30 mV; the third component, the N-component, was transient and was activated at voltages close to -20 mV. 3. A statistical analysis made on seventy-one experiments showed that the L-component was the most frequent (65% of the experiments), followed by the T- and finally the N- components (59 and 29% of the experiments, respectively). 4. The substitution of Ba2+ ions for Ca2+ ions greatly enhanced the L-component's amplitude (iBa/iCa = 4) while the N-component was unaffected and the T-component was reduced (iBa/iCa = 0.4). 5. A comparison of the voltage-dependent steady-state inactivation of the three components showed that the T-component was inactivated at -60 mV while the inactivation of the L- and N-components was complete at -25 and 0 mV, respectively. 6. A run-down effect was detected in some cells. The time stability of the L-component was lower than that of the T-component. The N-component seemed to be insensitive for at least 1 h. The results for the L- and T-components were obtained in cells which presented no run-down of the current or only a weak one. 7. Cd2+ ions (5 x 10(-5)M) completely blocked the long-lasting component (L-component) and slightly decreased the T-component. 8. Bay K 8644, a dihydropyridine agonist, enhanced the L-component at a concentration of 2.5 microM but decreased it for a higher concentration (5 microM). The T-component was decreased in a reversible way by 1 microM-Bay K 8644. Nifedipine, a well-known antagonist, blocked completely the L-component. This effect was reversed by the addition of Bay K 8644 to the perfusion medium. The T-component was also blocked by nifedipine, a result which is in keeping with the fact that Bay K 8644 has a weak effect on this current.
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PMID:Three components of the calcium current in cultured glomerulosa cells from rat adrenal gland. 247 2

1. Ionic currents associated with the invasion of an action potential into the motor nerve ending of the lizard, Anolis carolinensis, were measured with a focal extracellular electrode at several locations along the nerve ending. 2. These experimentally observed currents could be matched with computer simulations of action potential propagation into the nerve ending. They revealed that while Na+ channels are the major ionic current pathway in the heminode, K+ channels provide the major pathway in the terminal branches and boutons. 3. Calcium current in the presynaptic ending was unmasked by the application of tetraethylammonium (TEA). This current was blocked by: (a) cadmium, (b) omega-conotoxin GVIA and (c) nifedipine, but was unaffected by nickel at concentrations less than or equal to 100 microM. Nifedipine's action became more definitive when the duration of the action potential was greatly extended by pre-treatment with TEA. The effect of Bay K 8644 was inconsistent. 4. Transmitter release, as measured by postsynaptic current, had a pharmacological response profile similar to that of the Ca2+ current, with the exception that transmitter release was increased reliably and reversibly by Bay K 8644. 5. This pharmacological response profile is identical to that of the L type Ca2+ channel identified by Fox, Nowycky & Tsien (1987 alpha) in chick dorsal root ganglion neurones. We saw no evidence for more than a single type of Ca2+ channel in lizard motor nerve endings. 6. A calcium-activated K+ current IK(Ca) was revealed by application of 3,4-diaminopyridine (DAP), a delayed-rectifier K+ channel blocker. This K(Ca) current was blocked by TEA, charybdotoxin and by substitution of cobalt for extracellular calcium.
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PMID:Identification of ionic currents at presynaptic nerve endings of the lizard. 257 61

1. The adenosine analogue 2-chloroadenosine (CADO) reduced the duration of calcium-dependent action potentials (CAPs) in mouse dorsal root ganglion (DRG) neurones in culture, by reducing voltage-activated calcium conductance (Macdonald, Skerritt & Werz, 1986). Using the single-electrode voltage clamp technique, we recorded three calcium current components in these neurones, the transient low-threshold (T), transient high-threshold (N) and slowly inactivating high-threshold (L) currents, as described previously (Nowycky, Fox & Tsien, 1985; Gross & Macdonald, 1987). CADO (100 microM) had no effect on the isolated T and L currents. In contrast, CADO reduced calcium currents evoked at clamp potentials positive to -20 mV from holding potentials (Vh) near the resting membrane potential; under these conditions, the calcium current consisted primarily of N and L calcium current components. 2. This effect of CADO was not voltage dependent. CADO reduced the magnitude of the calcium current without affecting the voltage dependence of the calcium current-voltage relation. In addition, similar reductions of calcium current were observed when currents were evoked from Vh of -60 or -80 mV. 3. In order to determine if a guanine nucleotide-binding (G) protein was involved in the CADO effect on calcium current, cultures were pre-treated with pertussis toxin (PT) for at least four hours. PT (100 ng/ml) reduced or abolished the CADO-induced reduction of CAP duration and calcium current. 4. Since CADO inhibits adenylate cyclase through the PT-sensitive G protein, Gi, we compared the effects of CADO and 8-Br-adenosine 3',5'-cyclic-monophosphate (8-Br-cyclic AMP) on calcium current. The effect of 8-Br-cyclic AMP was voltage dependent, unlike that of CADO. 8-Br-cyclic AMP reduced calcium currents evoked from Vh = -65 mV, but had no effect on currents evoked from Vh = -85 mV. 5. We conclude that the adenosine agonist CADO reduced CAP duration in mouse DRG neurones by selectively reducing the N current component, and that the coupling between the adenosine receptor and the calcium channel required a PT-sensitive G protein. The CADO effect was unlikely, however, to be due to modulation of adenylate cyclase activity.
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PMID:2-Chloroadenosine reduces the N calcium current of cultured mouse sensory neurones in a pertussis toxin-sensitive manner. 261 35

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.
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PMID:Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution. 270 43

The Mr approximately 540,000 dimeric actin gelation protein, actin-binding protein (ABP), has previously been shown in human platelets to link actin to membrane glycoprotein Ib (GPIb) (Fox, J. E. B. (1985) J. Biol. Chem. 260, 11970-11977; Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., and Kunicki, T. J. (1985) J. Cell Biol. 100, 317-321). We have examined further the interaction between ABP and GPIb. Platelet extracts were depleted of ABP by precipitation with anti-ABP monoclonal antibodies (mAbs); in resulting precipitates, ABP monomer is complexed with GPIb in a 5:1 molar ratio. The ABP.GPIb complex is resistant to chaotropic solvents but dissociated by the ionic detergent, sodium dodecyl sulfate. Treatment of intact platelets with the ionophore A23187 activates a Ca2+-dependent protease which cleaves the Mr approximately 270,000 ABP subunit into three fragments of Mr 190,000, 100,000, and 90,000; the latter fragment is derived from the Mr 100,000 fragment. Anti-ABP mAbs coprecipitated GPIb with the Mr 100,000 and 90,000 fragments, but not with the Mr 190,000 fragment which contains the ABP self-association site. In the reciprocal experiment, anti-GPIb antibodies co-precipitated only the Mr 100,000 and 90,000 ABP fragments. Actin also co-precipitated with the Mr 100,000 and 90,000, but not with the Mr 190,000 ABP fragment. The anti-ABP mAb that precipitated the Mr 100,000-90,000 GPIb-binding ABP fragment recognizes a trypsin cleavage fragment of ABP that binds actin filaments in vitro. These findings establish that both the GPIb-binding site and actin-binding sites are in the same region of the ABP monomer. Because of the extended bipolar conformation of the ABP molecule, the data suggest that the GPIb.actin-binding region is located remote from the self-association, or dimerization, site of the ABP subunit.
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PMID:Localization of the domain of actin-binding protein that binds to membrane glycoprotein Ib and actin in human platelets. 313 34

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