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.     

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     

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.     

Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTP[S] , GDP and ATP for maximal activation were 0.1mM, 5M,1 mM and 1 mM respectively. The activation caused by 1mM ADP was lower. The enzyme was not activated by 1mM AMP, but significant activation was observed by the addition of 1mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A ablished the activation. There were synergic effects between ATP and GTP, ATP and PIP₂, but not between ATP and GTP[S] , or PIP₂ and GTP[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP₂ scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP₂ is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.     

Effects of G-protein probes on interactions between auxin efflux carriers and phytotropin receptors in zucchini (Cucurbita pepo L.) microsomal membranes

Microsomal vesicles prepared from etiolated hypocotyl tissue of zucchini (Cucurbita pepo L. cv. All Green Bush) exhibited saturable N-1-naphthylphthalamic acid ([³H]NPA) binding, NPA-stimulated association of indol-3yl-acetic acid ([³H]IAA), and saturable binding of guanosine 5-O-[3-thiotriphosphate] (GTP--[³⁵S]). These vesicles were used to test the possibility that NPA receptors might interact with IAA-anion efflux carriers by coupling through a GTP-binding protein (G-protein). Unlabelled GTP--S or guanosine 5-O-[2-thiodiphosphate] (GDP--S) had no effect on saturable NPA binding or on the NPA-stimulated association of IAA with microsomes. NPA did not affect saturable binding of GTP--[³⁵S] to microsomes, either in the presence or absence of saturating concentrations of unlabelled GTP--S or GDP. It is concluded that the occupancy of phytotropin receptors is not transduced to auxin efflux carriers by a GTP-binding protein.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Dopamine Is a Natural Inverse Agonist in the Lymnaea Nervous System

Investigation of [³⁵S]GTPS binding with G proteins in plasma membranes of the molluscan nervous system demonstrated the existence of an inverse agonism phenomenon for dopamine.     

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.     

Unisite Hydrolysis of [γ32P]ATP by Soluble Mitochondrial F1ATPase and Its Release by Excess ADP and ATP. Effect of Trifluoperazine

Some of the characteristics of unisite hydrolysis of [³²P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [³²P]ATP bound at the catalytic sites of F₁ under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [³²P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [³²P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [³²P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [³²P]ATP/³²Pi bound at the catalytic site and a 50% diminution in the rate of ³²Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F₁ in the sense that in a fraction of F₁ molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [³²P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.     

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/.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Transmembrane signaling through phospholipase C in human cortical membranes

Stimulation of phosphoinositide-specific phospholipase C (PLC) by carbachol, dopamine and serotonin was measured by supplying exogenous [³H]phosphatidylinositol 4,5-bisphosphate to membranes prepared from human cortex dissected and frozen at autopsy. Subjects with Alzheimer's disease, Parkinson's disease or schizophrenia were compared to age-matched controls with no known neurological disorders. Stimulation of PLC by the neurotransmitters was dependent on the presence of GTPS. Carbachol elicited the greatest stimulations of PLC followed by serotonin and then dopamine. The maximal stimulations of PLC evoked by a neurotransmitter were similar for the various categories of subjects except in Parkinson's patients, where dopamine failed to stimulate PLC beyond the activity attained with carbachol. In the presence of carbachol, the sensitivity of PLC to GTPS was significantly increased in Alzheimer's membranes, but not in age-matched controls or Parkinson's. Overall, the experiments demonstrate the feasibility for using the exogenous substrate assay to study the functionality of the phosphoinositide transmembrane signaling system in human brain.Abbreviations PLC phospholipase C - GTPS guanosine 3-O-thiotriphosphate - Gpp(NH)p 5-guanylyl-imidodiphosphate - DA dopamine - CCh earbachol - 5-HT serotonin - PIP₂ phosphatidylinositol 4,5-bisphosphate - PIP phosphatidylinositol 4-bisphosphate     

Cytochalasin B inhibits phosphoinositide hydrolysis in rat hippocampal slices

The effect of cytochalasin B on phosphoinositide (PI) hydrolysis was examined in rat hippocampal slices. Pretreatment of the slices with cytochalasin B caused a significant decrease in PI hydrolysis elicited by carbachol, norepinephrine, or by high K⁺. This effect was cytochalasin B dose- and time-dependent and was not mimicked by cytochalasin D, vinblastine, colchicine, or phloretin. In contrast, in [³H]inositol-prelabeled hippocampal membranes, cytochalasin B did not affect PI hydrolysis elicited by GTPS and GTPS plus carbachol. Similar result was obtained using the membranes prepared from the slices pretreated with cytochalasin B. The inhibitory effect of cytochalasin B on the carbachol-response was observed in SK-N-SH human neuroblastoma cells, but not in cultured rat astrocytes. These results indicate that cytochalasin B inhibits PI hydrolysis in neuron-specific manner and that its action may be an indirect cellular mechanism other than interaction with cytoskeleton elements.     

Detection of a 65 kDaras binding protein in rat and sheep brain cytosol using a chemical cross linking agent

The ability of aras protein to associate with proteins present in rat brain cytosolin vitro was investigated using chemical cross-linking agents and the¹²⁵I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [¹²⁵I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [¹²⁵I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [¹²⁵I] 85 kDa complex was inhibited by unlabelledras protein, GTP, GTPS, and GDP but not by ATPS and GMP.Chromatography of the cross-linked brain cytosol-[¹²⁵I]ras mixture on DEAE cellulose partially resolved the [¹²⁵I] 85 kDa complex from the [¹²⁵I]ras protein. The [¹²⁵I] 85 kDa complex (formed using ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [¹²⁵I]-labelledras. A substantial enrichment of the proportion of the [¹²⁵I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that thein vitro chemical cross-linking approach employed here has detected tworas binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDaras binding protein is aras regulatory orras effector protein which has not so far been characterised is briefly discussed.Abbreviations DSS disuccinimidyl suberate - EGS ethyleneglycolbis (succinimidylsuccinate) - GTPS guanosine 5-[-thio] triphosphate - ATPS adenosine 5-[-thio] triphosphate     

Hexachlorocyclohexanes affect the arachidonic acid release from phosphatidylinositol but not from other phospholipid classes in tubular cell cultures

Gamma- and delta-isomers of hexachlorocyclohexane caused marked decreases in the levels of radioactive phospholipids, and increases in the levels of [³H]arachidonate incorporated into free fatty acids in rat renal tubular cells. The increased radioactivity of free fatty acids arises from the decrease of [³H]arachidonate incorporated into phosphatidylinositol, but not into phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. This fact suggests that phosphatidylinositol can be broken down to the fatty acid from the sn-2 position and lysophospholipid by a phospholipase activity increased by hexachlorocyclohexanes. The observed specific toxicant action could be achieved in two ways: (a) operating upon a specific phospholipase A₂ that acts on phosphatidylinositol, but not on other phospholipids as substrates and/or (b) involving substrate-phospholipase A₂ interactions. Interestingly, the observed effect of the -isomer was more pronounced than that of the -one.     

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.     

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     

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.     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.