Actions of Ca2+ channel ligands depend on G-proteins. Ca2+ agonists/antagonists and GTP.
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Animals↗
Biomedical subjects
R H Scott
Publications and source records attributed to R H Scott.
Modulation of divalent cation-activated chloride ion currents.
1. Voltage-sensitive calcium channel currents carried by Ca2+ (ICa) or Ba2+ (IBa) were followed by tail currents carried by Cl- ions in approximately 45% of cultured dorsal root ganglion neurones. 2. Extracellular application of (-)-baclofen (100 microM) inhibited IBa and ICl(Ba). Bay K 8644 (5 microM) potentiated both currents. 3. Intracellular GTP-gamma-S increased the proportion of neurones in which ICl(Ba) was recorded. In addition, the activation by GTP-gamma-S of a pertussis toxin-sensitive GTP binding (G)-protein resulted in a steady increase in the Cl- tail current with time, despite a concurrent reduction in IBa. 4. Extracellular application of 10mM caffeine selectively reduced ICl(Ba) without significant change in IBa. When Ca2+ was the charge carrier, caffeine had little effect on ICl(Ca), and increased the inactivation of ICa. 5. We conclude that, in addition to being regulated by divalent cation entry through Ca2+ channels, the Cl- current is also regulated by G-protein activation. The mechanism of activation of ICl(Ba) may involve Ca2+ release from intracellular stores.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Influence of heat treatment on FVIII:C recovery from freeze dried cryoprecipitate.
A standard lyophilised triple cryoprecipitate preparation, stabilised by the addition of Synthamin 17, was heat treated at 60 degrees C for 48 hours. The total protein content, factor VIII concentration, and factor VIII recovery were not affected by the heat treatment procedure. Heat treatment did not influence the reconstitution characteristics of the freeze dried preparation and there were no side effects during or after administration. The mean in vivo rise of factor VIII from infused heat treated triple cryoprecipitate was 2.5 (SD 0.9)%/unit/kg with a half life of 13.1 (3.1) hours. These results compare favourably with those obtained using non-heated triple cryoprecipitate. Cryoprecipitate can be heat treated without adversely influencing factor VIII recovery, and the ability to prepare a heat treated cryoprecipitate means that a small pool high yield factor VIII preparation can again be used in routine clinical practice.
Adolescent↗
Pharmacology of GABA receptors on skeletal muscle fibres of the locust (Schistocerca gregaria).
GABA and the trans isomer of 4-aminocrotonic acid are equally potent at inducing increases in Cl- conductance when applied to distal extensor tibia muscle fibres of the locust (Schistocerca gregaria). beta-Alanine, norvaline, glycine and norleucine induced conductance increases of less than 5% of GABA responses. C9 and meso-di-GABA did not alter input conductance in a manner consistent with actions on a GABA receptor Cl- channel complex. Picrotoxin and anisatin were equally potent GABA antagonists, however bicuculline and penicillin G did not reduce GABA-induced changes in input conductance. Pentobarbitone, in addition to inducing an increase in K+ conductance, potentiated GABA-induced increases in Cl- permeability.
Amino Acids↗
Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides.
1. The effect of intracellular application of the hydrolysis-resistant GTP and GDP analogues, guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S), and guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) has been examined on voltage-activated calcium-channel currents and the ability of the gamma-aminobutyric acid B agonist baclofen to inhibit them, in cultured rat dorsal root ganglion (d.r.g.) neurones. 2. Under control conditions, the calcium-channel current, recorded using the whole-cell patch technique with Ba2+ rather than Ca2+ as the permeant divalent cation, consists of an inactivating and a sustained current. In the presence of 500 microM-GTP-gamma-S included in the patch pipette, the calcium-channel current was activated more slowly and was largely non-inactivating during the 100 ms depolarization voltage step. The effects of GTP-gamma-S were abolished by pre-treatment of cells with pertussis toxin. 3. The calcium-channel current recorded in the presence of 500 microM-GDP-beta-S had a more marked transient component than the control calcium-channel current. The proportion of transient calcium-channel current in the presence of GDP-beta-S was not reduced in Na+-free medium. 4. No statistically significant effects of GTP-gamma-S and GDP-beta-S were observed on the calcium-activated potassium current IK(Ca), the transient outward potassium current activated in Ca2+-free medium, or on the inwardly rectifying current (Ih) activated by hyperpolarization. 5. GTP-gamma-S increased the ability of baclofen to inhibit calcium-channel currents, whereas this was decreased by GDP-beta-S and by pre-treatment of cells with pertussis toxin. The half-maximal effective dose (EC50) for baclofen was 2 microM in the presence of GTP-gamma-S, 15 microM for control and 50 microM in the presence of GDP-beta-S. Comparable results were obtained using a single concentration of the adenosine agonist 2-chloroadenosine (2-CA, 0.05 microM) to inhibit calcium-channel currents; its effect was significantly increased by GTP-gamma-S and reduced by GDP-beta-S. 6. The ability of baclofen to inhibit calcium-channel currents was not affected by 1 microM-forskolin or 50 microM-intracellular cyclic AMP. 7. It is concluded that calcium channels in d.r.g.s are associated with a nucleotide binding protein, and that this mediates the effect of baclofen and 2-CA on calcium-channel currents. The ability of GTP-gamma-S to inhibit the transient component of calcium-channel currents in the absence of agonist may represent a means of differentially regulating calcium-channel activity.
Action Potentials↗
Regulation of calcium currents by a GTP analogue: potentiation of (-)-baclofen-mediated inhibition.
Voltage-activated calcium currents were recorded in cultured rat dorsal root ganglion neurones using the whole cell clamp technique. The effect of inclusion in the patch pipette of the non-hydrolysable guanine nucleotide analogues, guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) and guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) was examined both on ICa itself and on the ability of the GABAB agonist baclofen to inhibit ICa. In the absence of either guanine nucleotide, the maximum ICa recorded at between -20 and 0 mV consists of two components, a rapid transient and a non- or slowly inactivating current. Baclofen (50 microM) inhibited peak ICa by 37.5 +/- 4.7%. In the presence of 500 microM GTP-gamma-S the inactivating calcium current was largely absent and the effect of 50 microM baclofen was increased to a 78.1 +/- 2.3% inhibition. GDP-beta-S had opposite effects to GTP-gamma-S. The results suggest that the calcium channels in these neurones are associated with a GTP binding protein which regulates the calcium currents and mediates the inhibitory effect of neurotransmitters.
Animals↗
Anion selectivity of gamma-aminobutyric acid (GABA) 22,23-dihydroavermectin B1a (DHAVM)-induced conductance changes on locust muscle.
Input conductance was recorded from extensor tibia muscle fibres of the locust Schistocerca gregaria. gamma-Aminobutyric acid (GABA; 2.5 X 10(-5) M to 5 X 10(-3) M) and 22,23-dihydroavermectin B1a (DHAVM; 0.01 micrograms/ml, 1.2 X 10(-8) M to 1.0 microgram/ml, 1.2 X 10(-6) M) induced dose-dependent increases in chloride (Cl-) ion permeability on distal muscle fibres. Changing the anion content (Br-, or I- substituted for Cl-) of the saline perfusing the locust muscle fibres induced additional conductance and small hyperpolarizations of the membrane potential. The predominant anion permeability sequences for conductance increases induced by GABA and DHAVM were I- greater than Br- greater than Cl- (n = 5) and Cl- greater than Br- greater than I- (n = 12), respectively. These findings suggest that the anion channel activated by GABA has low selectivity. The irreversible increase in conductance induced by DHAVM appears to involve an ion channel with greater selectivity for Cl-. However, DHAVM-induced changes in filtering properties of a single type of Cl- channel are not precluded. This is consistent with DHAVM action not being dependent on GABA sensitivity of the locust muscle fibres.
Animals↗
Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (-)-baclofen.
Voltage-dependent inward calcium currents (ICa) activated in cultured rat dorsal root ganglion neurones were reversibly reduced in a dose-dependent manner by (-)-baclofen (10 microM to 100 microM). Baclofen (100 microM) reduced the calcium-dependent slow outward potassium current (IK(Ca)). This current was abolished in calcium-free medium and by 300 microM cadmium chloride. The action of baclofen on IK(Ca) was reduced when the calcium concentration in the medium was increased from 5 mM to 30 mM. The calcium independent fast transient voltage-dependent outward current (IK(Vt] was also reduced by baclofen; this effect remained present when Ca2+-free medium was used to prevent contamination by IK(Ca). 4-Aminopyridine (500 microM) reduced IK(Vt) and induced a small increase in ICa. The action of baclofen on ICa was partially antagonized by 4-aminopyridine. GABAB receptor-mediated inhibition of ICa in cultured rat dorsal root ganglion neurones involves a direct mechanism rather than resulting indirectly from an increase in the residual outward potassium currents activated by depolarization. The reduction in ICa by baclofen was variable and dependent on the amplitude of control ICa, larger currents being more resistant to the baclofen-induced inhibition.
4-Aminopyridine↗
Calcium-dependent currents in cultured rat dorsal root ganglion neurones are inhibited by an adenosine analogue.
Dorsal root ganglion neurones from 2-day-old rats were grown in dissociated culture. The effect of the adenosine analogue 2-chloroadenosine (2-CA) was investigated on action potential duration and on Ca2+ current (ICa) activation. 2-CA (0.5 microM) shortened both control action potentials and those prolonged by tetraethylammonium (TEA), Ba2+, or intracellular Cs+. This effect was prevented by two adenosine antagonists isobutylmethylxanthine (IBMX, 1-2 mM) and 8-phenyltheophylline (8-PT, 2.5 microM). The inward current, ICa, recorded using the 'whole-cell' patch-clamp technique in medium containing 2.5 microM-tetrodotoxin, 25 mM-TEA and 2.5 mM-Ba2+ was reduced by 2-CA (0.05 microM). The activation of ICa was decreased, but its reversal potential was unchanged. The effect of 2-CA was antagonized by IBMX (1 mM) and 8-PT (1 microM). 2-CA also reduced the large inward tail currents which occurred at the termination of the depolarizing voltage step command in a proportion of neurones. Brief application of 2-CA (0.05 microM) did not affect the inward holding current required to maintain the cells at -80 mV. In the presence of TTX (2.5 microM) and Ca2+ (5 mM), 2-CA decreased the activation of outward K+ currents caused by 5 s depolarizing voltage commands from -80 mV or -40 mV. The GABAB agonist (-)-baclofen (50-100 microM) also shortened the action potential duration and reduced ICa. 8-PT (1 microM) did not prevent the effect of baclofen on ICa. It is concluded that in cultured rat dorsal root ganglion neurones 2-CA reduces ICa activation by a direct effect on an A1 adenosine receptor.
1-Methyl-3-isobutylxanthine↗
Actions of dihydroavermectin B1a on insect muscle.
Muscle bundle 33 of the locust (Schistocerca gregaria) extensor tibiae muscle which is sensitive to gamma-aminobutyric acid (GABA) and receives inhibitory innervation, exhibited both reversible and irreversible responses to dihydroavermectin B1a (DHAVM). These responses involved increases in C1- permeability. DHAVM (0.000075-0.0075 microgram ml-1) induced reversible dose-dependent increases in C1- permeability and partially blocked GABA-induced C1- conductance. These effects appear to be due to an interaction of DHAVM with the GABA receptor-C1- ion channel complex. DHAVM (0.01-1.0 microgram ml-1) induced an irreversible increase in C1- conductance which continued to rise after DHAVM application was stopped. At these concentrations DHAVM potentiated GABA-induced C1- conductances which were in turn reduced by microperfusion of DHAVM (0.01-1.0 microgram ml-1) during bath application of GABA. DHAVM (0.0001-1.0 microgram ml-1) induced only irreversible increases in C1- conductance when applied to fast muscle bundles (21-26) of the locust extensor tibiae muscle, which are GABA-insensitive and have no inhibitory innervation. The actions of DHAVM on locust muscle appear to involve more than one site. Reversible actions of DHAVM appear to be related to GABA sensitivity and may involve the GABA receptor-ionophore complex. This is unlikely to be the site of action for the irreversible increases in C1- conductance caused by DHAVM.
Animals↗
Preparation of leucocyte poor blood for transfusion.
Three methods for the preparation of leucocyte poor red cells for transfusion have been evaluated. A new filtration (IMUGARD) method produced an easily prepared high quality leucocyte-poor preparation but which was not suitable for prolonged storage. A simple centrifugation method was suitable for the preparation of a sterile long life leucocyte-poor preparation and could be used for the majority of patients. A dextran sedimentation method was efficient in leucocyte removal but with the loss of a significant proportion of red cells.
Blood Sedimentation↗
Characterization of subunit structural alterations which occur during purification of nitrate reductase from Escherichia coli.
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Bacterial Proteins↗
Naloxone and shuttlebox self-stimulation in the rat.
Rats self timed electrical brain stimulation on and off periods in a shuttlebox. Electrodes for self-stimulation were located either in the lateral hypothalamic area (LHA) or the periaqueductal gray (PAG). Doses of the narcotic antagonist, naloxone, were administered intraperitoneally immediately prior to self-stimulation testing. Doses of 1.0, 5.0, 10.0 or 50.0 mg/kg failed to alter shuttlebox self-stimulation behavior. These results are inconsistent with one lever-press self-stimulation study employing PAG electrodes [3], but agree with other studies using LHA electrodes [9, 15, 21, 24). Possible reasons for the discrepancy are suggested.
Animals↗
In vitro incorporation of molybdate into demolybdoproteins in Escherichia coli.
When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.
Aldehyde Oxidoreductases↗
Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.
Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.
Amino Acids↗
Role of L-proline in the biosynthesis of prodigiosin.
Nonproliferating cells of Serratia marcescens, wild-type strain Nima, synthesized the pigment, prodigiosin, when saline suspensions were incubated with aeration at 27 degrees C in the presence of proline or alanine. Mutants PutS1 and PutS2 derived from strain Nima formed prodigiosin from alanine, but not from proline, unless alanine also was added. Strain Nima utilized proline as a sole source of carbon and of nitrogen for growth, whereas Put mutants did not. Investigation of enzymes degrading proline showed that the wild-type strain contained proline oxidase, which was absent in Put mutants. The wild type, as well as the mutants, utilized alanine as the sole source of carbon and nitrogen for growth. Although nonproliferating cells of Put mutants failed to synthesize prodigiosin from proline, addition of L-[U-14C]proline to suspensions metabolizing and synthesizing the pigment because of addition of alanine resulted in the incorporation of radioactive label into prodigiosin, as well as into cellular protein. Since Put mutants could not catabolize proline, the incorporation of [14C]proline into the prodigiosin molecule indicated that proline was incorporated directly into the pigment.
Alanine↗
Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli.
When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited. Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway. Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels. When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present. Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels. This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol. The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels. Cytochrome b1 was again associated with this band. The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels. Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position. Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells. Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes. Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.
Aldehyde Oxidoreductases↗