Regulation of Phospholipase C Activity by Calcium Ions and Guanine Nucleotide in the Normoxic Cat Carotid Body

The carotid bodies (CB) are a paired chemoreceptor organ located at the bifurcation of the common carotid arteries. High O₂ tension suppresses while low tension activates afferent carotid chemoreceptor activity and the chemoreflex ventilatory response in the cat. The intracellular mechanism of chemotransduction is till now unknown. Previously we have shown different activities of phospholipase C (PLC) in normoxic, hypoxic and hyperoxic cat carotid body. Now we have addressed the question whether calcium ions and G-protein could be regulators of the formation of lipid derived messenger molecules in the cat carotid body. To answer this question, the PLC acting against [³H] inositol-phosphatidylinositol (PtdIns) and [³H] inositol-phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P₂] in the cat CB were investigated using labelled phospholipids as a source of the substrate. CB homogenate was used as a source of the enzyme. The results indicate that PLC acting on PtdIns is Ca²⁺-dependent, in contrary to that acting on PtdIns(4,5)P₂ which remains active in the presence of 10 mM EGTA. PtdIns(4,5)P₂-PLC is stimulated by GTPS. In the presence of Ca²⁺, GTPS has a synergistic stimulatory effect. PLC acting on PtdIns is not activated by GTPS. In the presence of calcium ions dopamine and a nonhydrozylable analogue of acetylocholine, carbachol, have a small stimulatory effect of about 30 % on PLC acting on PtdIns(4,5)P₂. GTPS enhances this effect. These results allow us to suggest that there are two pathways of phosphoinositides degradation in the CB, one of them is regulated by calcium ions/PtdIns-PLC/, the other one by G-protein/PtdIns(4,5)P₂-PLC/.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate     

Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat

Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [³⁵S]GTPS with a relatively high affinity (K_d10nM). The binding was saturable and specific as it was indicated by the displacement of bound [³⁵S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [³⁵S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [³⁵S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [³⁵S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - G protein hetero-trimeric GTP binding protein with , , subunits - G_n protein GTP binding protein detected on nitrocellulose blots - GTPS guanosine 5-[-thio]triphosphate - IAA 3-indoleacetic acid - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca²⁺ (EC₅₀=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca²⁺ concentrations. At optimal Ca²⁺ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A₂. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca²⁺ dependent effects, (ii) phospholipase C was not activated in the absence of Ca²⁺ and insulin secretion due to the phorbol ester TPA displayed a different Ca²⁺ dependency, (iii) arachidonic acid did not elicit Ca²⁺ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca²⁺ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP₃ inositol trisphosphate - Ptd-InsP₂ phosphatidylinositol 4,5-bisphosphate - GTPS guanosine 5-(3-O-thio)triphosphate - GDPS guanosine 5-(2-O-thio)diphosphate - Gpp(NH)p guanyl-5-yl imidodiphosphate - TPA 12-O-tetradecanoylphorbol-13-acetate - OAG 1-oleoyl-2-acetylglycerol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid - DAG diacylglycerol - [Ca²⁺]_i cytosolic free Ca²⁺ concentration     

Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Action of a nonhydrolyzable guanosine triphosphate analog on modulation of calcium current by dopamine in Lymnaea stagnalis neuron

Dependence of the blocking effect of dopamine on the calcium current on guanosine triphosphate (GTP) was investigated on dialyzed neurons ofLymnaea stagnalis. Against the background of the effect of the nonhydrolyzable GTP analog guanosine-5-0-3-thiotriphosphate (GTPS) (10^–4 M) marked residual blocking of the calcium current was observed during rinsing out of dopamine, and this was potentiated in the case of repeated application of the hormone. It is suggested that the receptor-mediated effect of GTPS on the calcium current is effected through activation of phospholipase C.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 43–48, January–February, 1991.     

Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. InMyo-[³H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [³H]inositol phosphates ([³H]IPs) accumulation (EC₅₀ of 3±1 M). The M₁-selective antagonist pirenzepine (PRZ), the M₂-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) and the M₃-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [³]inositol phosphates accumulation with the following order of potency: p-F-HHSiD>PRZ>AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTP[S] elicited a concentration-dependent increase in [³H]inositol phosphates accumulation. The concentration-response curve for GTP[S] was shifted to the left when cells were incubated with 1 M carbachol. The [³H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 M GTP[S] and 1 M carbachol to permeabilized cells was significantly decreased (78.28±18.23% inhibition) when cells were preincubated for 5 min with 0.1 mM GDP[S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [³H]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 M final concentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 M isoproterenol was abolished by pertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabbit caecum, the muscarinic receptor stimulation of [³H]inositol phsophates accumulation is mediated by M₃ subtype receptors coupled to a pertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M₂ subtype receptors coupled to a pertussis toxin-sensitive GTP-binding protein G_i.     

Characterization of specific calcitonin gene related peptide receptors present in hamster pancreatic β cells

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of¹²⁵I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound¹²⁵I-[His]hCGRP I could be induced in the presence of 1 M chicken CGRP (cCGRP). The specific¹²⁵I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC₅₀) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0–2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of¹²⁵I-[His]hCGRP I and sCT was only effective at a high concentration (1 M). Binding of¹²⁵I-[His]hCGRP I was dose dependently inhibited by guanosine-5-O-(3-thiotriphosphate) or (GTPS) and a 70% reduction of binding was obtained with 0.1 mM GTPS. The IC₅₀ value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTPS. Human CGRP I and cCGRP at 2.5 M did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 M) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.Abbreviations CGRP calcitonin gene-related peptide - IAPP islet amyloid polypeptide - IC₅₀ half-maximal inhibitory concentration - GTPS guanosine-5-O-(3-thiotriphosphate) - ¹²⁵I [His]hCGRP I, (2[¹²⁵I]iodohistidyl¹⁰) human CGRP I     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Utilization and degradation of lindane by soil microorganisms

Of 147 microorganisms isolated from a loamy sand, 71 showed good growth with lindane (-1,2,3,4,5,6-hexachlorocyclohexane) and produced chloride in an aqueous medium. Thirteen soil microorganisms were selected to study the utilization of lindane. Lindane was metabolized by the microbes to -2,3,4,5,6-pentachloro-1-cyclohexene (-PCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), and pentachlorobenzene (PCB). Cells of Pseudomonas sp. No. 62 grown on lindane simultaneously adapted to -PCCH, -TCCH, -TCCH, -TCCH, PCB, 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB). The bacteria degraded each of these chemicals at least partially as indicated by an increased rate of oxygen consumption.Abbreviations Lindane -1,2,3,4,5,6-hexachlorocyclohexane - -PCCH -2,3,4,5,6-pentachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - PCB pentachlorobenzene - 1,2,3,4-TCB 1,2,3,4-tetrachlorobenzene - 1,2,3,5-TCB 1,2,3,5-tetrachlorobenzene - 1,2,4,5-TCB 1,2,4,5-tetrachlorobenzene - 1,2,3-tCB 1,2,3-trichlorobenzene - 1,2,4-tCB 1,2,4-trichlorobenzene - 1,3,5-tCB 1,3,5-trichlorobenzene - 1,2-DCB 1,2-dichlorobenzene - 1,3-DCB 1,3-dichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene Contribution No. 631, Research Institute, Agriculture Canada, University Sub Post Office, London, Ontario N6A 5B7     

Regional Activities of Phospholipase C after Experimental Brain Injury in the Rat

Regional activities of phosphoinositide-specific phospholipase C (PLC) were measured after lateral fluid percussion (FP) brain injury in rats. The activity of PLC on phosphatidylinositol 4,5-bisphosphate (PIP₂) in the rat cortex required calcium, and at 45 M concentration it increased PLC activity by about ten-fold. The activity of PLC was significantly increased in the cytosol fraction in the injured (left) cortex (IC) at 5 min, 30 min and 120 min after brain injury. However, in the same site, increases were observed in the membrane fraction only at 5 min after brain injury. In both the contralateral (right) cortex (CC) and ipsilateral hippocampus (IH), the activity of PLC was increased in the cytosol only at 5 min after brain injury. These results suggest that increased activity of PLC may contribute to increases in levels of cellular diacylglycerol and inositol trisphosphate in the IC (the greatest site of injury), and to a smaller extent in the IH and CC, after lateral FP brain injury. It is likely that this increased PLC activity is caused by alteration in either the levels or activities of one or more of its isozymes (PLC, PLC, and PLC) after FP brain injury.     

Polyphosphoinositide phospholipase C and evidence for inositol-phosphate-hydrolysing activities in the plasma-membrane fraction from light-grown wheat (Triticum aestivum L.) leaves

The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca²⁺ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca²⁺ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP₂) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca²⁺ the enzyme clearly favoured PIP. In addition, the PIP₂-PLC activity was increased by Mg²⁺ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P₃ was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP₂-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC deoxycholate - IDPase inosine 5-diphosphatase - InsP_s inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e. - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PLC phospholipase C This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance.     

Regulation of G protein function: Implications for heart disease

Heterotrimeric GTP-binding and -hydrolyzing proteins (G proteins) link members of a family of seven-helix transmembrane receptors (G protein-coupled receptors, GPCR) to intracellular effectors. The coupling mechanism involves the G protein completing a cycle of activation, dissociation into and subunits, deactivation, and reassociation. At the center of this cycle is the subunit, in which activation by GPCR, GTPase activity, and regulation of effector are combined. Whereas G's functional domains and residues had already been inferred from mutagenesis studies, the recent solution of the crystal structure has elucidated the structural basis of subunit function. It is now clear that an irregularity in any GPCR pathway component could cause a physiological defect. This is confirmed by the identification of mutations in GPCR and G's in various human diseases. Although several cardiomyopathies are associated with abnormal GPCR function, mutations are unlikely in these disorders. The last few years, other aspects of G protein function have moved into focus: e.g. posttranslational modifications; effector regulation by subunits; GTPase activating protein (GAP) activity of effectors; G protein expression levels etc. When comparing the regulation of G protein functional activity in cAMP and in inositol phosphate generating pathways, an extrapolation can be made to data on the status of these pathways in some cardiovascular diseases.Abbreviations AC adenylate cyclase - GPCR G protein-coupled receptor - PLC phospholipase C - GAP GTPase activating protein - PTX pertussis toxin - Ptdins(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - CCh carbachol     

Reversal of defective G-proteins and adenylyl cyclase/cAMP signal transduction in diabetic rats by vanadyl sulphate therapy

Vanadium salts exhibit a wide variety of insulinomimetic effects. In the present studies, we have examined the modulation of G-protein levels and adenylyl cyclase activity in the liver of streptozotocin-induced chronic diabetic rats (STZD) by vanadyl sulfate treatment and compared it with that of insulin. The basal enzyme activity, as well as the stimulatory effects of guanine nucleotides, glucagon, N-Ethylcarboxamideadenosine (NECA), isoproterenol, forskolin and sodium fluoride (NaF) on adenylyl cyclase were significantly increased in STZ-D rat liver as compared to control. In addition, the levels of stimulatory (Gs) as well as inhibitory (Gi_-2 and Gi_-3) as determined by immunoblotting techniques were also significantly higher in the STZ-D rat liver, however, the inhibitory effects of oxotremorine and low concentration of GTPS on adenylyl cyclase were not different in the two groups. Vanadyl sulfate and insulin treatments restored the augmented basal enzyme activity, the stimulations exerted by stimulatory inputs on adenylyl cyclase and the G-protein levels to various degrees, however, vanadyl sulfate was more effective than insulin. In addition, unlike vanadyl sulfate, insulin was unable to improve the stimulation exerted by glucagon and isoproterenol on adenylyl cyclase activity in STZD rats. These results suggest that vanadyl sulfate mimics the effects of insulin to restore the defective levels of G-proteins and adenylyl cyclase activity. From these results it may be suggested that one of the mechanisms by which vanadyl sulfate improves the glucose homeostasis in STZ-D rats may be through its ability to modulate the levels of G-proteins and adenylyl cyclase signal transduction system.Abbreviations NECA N-ethylcarboxamideadenosine - Iso Isoproterenol - Glu Glucagon - FSK forskolin - GTPS guanosine 5-[-thio]triphosphate - Gs stimulatory guanine nucleotide regulatory protein - Gi inhibitory guanine nucleotide regulatory protein - STZ streptozotocin This work was supported by grants from Medical Research Council and Canadian Diabetes Association.     

Characterization of phosphoinositide hydrolysis products induced by hexachlorocyclohexane isomers in rat brain cortex

Water-soluble inositol metabolites were separated by anion-exchange chromatogrphy in order to determine whether or not -hexachlorocyclohexane (-HCH, lindane) and related compounds affect phosphatidylinositol hydrolysis in rat brain cortex slices. Hydrolysis was increased by -and -HCH, while - and -HCH were inactive. Muscarinic receptor stimulation of rat cortical slices with carbachol increases inositol phosphates formation. The combined effect of carbachol and the hexachlorocyclohexane isomers together were approximately equal to the sum of the effect of each one separately. The results suggest that lindane stimulates phosphoinositide phospholipase C and/or inhibits the phosphases implicated in dephosphorylation of inositol phosphates.     

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

The Arabidopsis G subunit, GP1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNA^Arg _AGA/AGG. Isolation of the recombinant GP1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPS withan apparent K_d in the nM range. GTPS binding wasstimulated two-fold in the presence of Zn²⁺ compared with that inthe presence of Mg²⁺, Mn²⁺ or Ca²⁺.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPS,guanosine- 5-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGP1, recombinant GP1     

Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: Characterization using [32P]3′-O-(4-benzoyl)benzoyl ATP,a photoaffinity label

3-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [-³²P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of ³²P incorporation in both proteins on [-³²P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca²⁺ or Mg²⁺. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP ÃDP>GTPS>ADP S, UTP, 2MeSATP, AMP-PNP >AMP-PCP>AMP>adenosine; for p96 it is: ADPADP S ATP AMP-PCP, AMP-PNP>GTPS AMP>2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADPS, UTP 2MeSATP, GTPS, AMP-PNP, ATPADP>AMPPCP>adenosine>AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P₂ purinoceptor. On the other hand, p96 may represent a P₂ purinoceptor or an ectonucleotidase.This work was supported by grants (to Z.P. and to V.S-B.) from the Fund for Basic Research administered by the Israel Academy of Science and Humanities.     

Dopamine Release Evoked by Beta Scorpion Toxin,Tityus Gamma,in Prefrontal Cortical Slices is Mediated by Intracellular Calcium Stores

1. We have investigated the effect of tityus gamma (TiTX ) scorpion toxin on the release of [³H] dopamine in rat brain prefrontal cortical slices. The stimulatory effect of TiTX on the release of [³H]dopamine was dose/time-dependent with an EC₅₀ of 0.01M.2. Tetrodotoxin blocked the TiTX -induced release of [³H]dopamine, indicating the dependency for Na⁺ channels.3. EGTA had no effect on the TiTX -induced release of [³H]dopamine, indicating the process is independent of extracellular calcium. Release of [³H]dopamine evoked by TiTX was inhibited by 57% by BAPTA, a chelator of intracellular calcium.4. Xestospongin and 2-APB, putative blockers of IP₃-sensitive release of intracellular calcium stores, caused an equal and significant inhibition of 24% of the TiTX -induced release of [³H]dopamine, while the slight inhibition evoked by dantrolene, a putative blocker of ryanodine-sensitive calcium store was not significant.5. Nomifensine and ascorbic acid, blockers of dopamine transporter (DAT), caused an inhibition of 27 and 29%, respectively, on the toxin-induced release of [³H]dopamine suggesting that most of the TiTX -induced release of dopamine is not due to the reversal of Na⁺ gradient.6. In conclusion the majority of the TiTX -induced release of [³H]dopamine is exocytotic and mobilizes calcium from the intracellular IP₃-sensitive calcium stores.     

Studies on the iodination of aras protein and the detection ofras polymers

Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greates incorporation of radioactivity with minimal modification of theras protein. Upon treatment of theras protein with [¹²⁵I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [¹²⁵I]ras was 6×10⁶ cpm/pmol totalras protein. Iondination did not alter the biological activity of theras protein as judged by its ability to bind GTPS and induced maturation ofXenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI ofras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. Theras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involvingras. Treatment of iodinatedras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purifiedras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.Abbreviations DSS Disuccinimidyl Suberate - GTPS Guanosine 5-[-thio] triphosphate - ATPS Adenosine 5[-thio] Triphosphate