scholarly journals Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex

1985 ◽  
Vol 232 (2) ◽  
pp. 501-504 ◽  
Author(s):  
V Hausleithner ◽  
M Freissmuth ◽  
W Schütz
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Radioligand Binding ◽  
Rank Order ◽  
Binding Studies ◽  
Gtp Hydrolysis ◽  
Sequence Of Events ◽  
Competition Binding ◽  
Cortical Membranes ◽  
Stimulation Of

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.

Positive coupling of beta-like adrenergic receptors with adenylate cyclase in the cnidarian Renilla koellikeri

1993 ◽  
Vol 182 (1) ◽  
pp. 131-146 ◽  
Author(s):  
E. W. Awad ◽  
M. Anctil
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Radioligand Binding ◽  
Guanine Nucleotides ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Binding Studies ◽  
Beta Adrenergic ◽  
Renilla Koellikeri ◽  
Stimulation Of

Coupling of the previously characterized beta1- and beta2-like adrenoceptors in the sea pansy Renilla koellikeri with adenylate cyclase was examined in membrane preparations from this cnidarian. Adenylate cyclase activity was stimulated by several guanine nucleotides, such as GTP, Gpp(NH)p and GTPgammaS. Fluoride ions and cholera toxin greatly enhanced the enzyme activity, whereas forskolin had no effect on basal or isoproterenol-induced stimulation of the enzyme. The stimulation of adenylate cyclase activity by several beta-adrenergic agonists in different parts of the animal reflected a positive coupling with the beta2- and beta1-like adrenoceptors in autozooid and peduncle tissues, respectively. In addition, isoproterenol-induced stimulation of adenylate cyclase activity was dependent on guanine nucleotides, suggesting coupling mediated by a G protein. The pharmacological profile of various antagonists on isoproterenol-sensitive adenylate cyclase in autozooid and peduncle tissues matched that of previous radioligand binding studies. Isoproterenol-induced stimulation of adenylate cyclase activity in rachidial tissues was partially inhibited by trifluoperazine of (+/−)CGP12177 and was completely blocked in the presence of both antagonists. This suggests that coupling of the enzyme occurs with beta1- and beta2-like adrenoceptors, both being present in the rachis. Serotonin and dopamine were also found to stimulate adenylate cyclase activity. Their stimulatory effect was additive to isoproterenol-induced activation, suggesting the presence of dopaminergic and serotonergic receptors in the tissues of the sea pansy. Along with the data presented previously on beta-adrenergic binding, this study suggests that elements of receptor-dependent G protein signal transduction originated early in invertebrate evolution.

The effect of diazepam on adenosine uptake and adenosine-stimuiated adenylate cyclase in guinea-pig brain

1982 ◽  
Vol 60 (3) ◽  
pp. 302-307 ◽  
Author(s):  
M. J. York ◽  
L. P. Davies
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Adenosine Deaminase ◽  
Small Component ◽  
Extracellular Adenosine ◽  
Pig Brain ◽  
Adenosine Uptake ◽  
Adenosine Antagonist ◽  
Guinea Pig Brain ◽  
Stimulation Of

We have used the adenosine-stimulated adenylate cyclase of guinea-pig brain to examine the potency of diazepam as an adenosine uptake inhibitor. Diazepam at concentrations in the range 10–500 μM stimulates the production of cAMP in incubated slices of guinea-pig cerebral cortex, with maximal fivefold stimulations over basal levels by 200 μM diazepam. The increases can be largely (but not completely) blocked by the adenosine antagonist theophylline or by addition of excess adenosine deaminase to the system. It appears that the stimulation of cAMP production is due to a blockade of adenosine uptake which results in an increase in extracellular adenosine and concomitant activation of the adenosine receptor coupled to adenylate cyclase. Since the cAMP response to standard adenosine uptake blockers (dipyridamole, dilazep) can be completely blocked by theophylline or adenosine deaminase, a small component of the diazepam response cannot be explained by an adenosine effect. The concentration of diazepam at which the first significant cAMP increase occurs is 10 μM or slightly lower. This is significantly higher than the concentration of diazepam needed to saturate the pharmacologically characterized central nervous system receptors for benzodiazepines.

scholarly journals PAR recognition by multiple reader domains of PARP1 allosterically regulates the DNA-dependent activities and independently stimulates the catalytic activity of PARP1

2021 ◽  
Author(s):  
Waghela Deeksha ◽  
Suman Abhishek ◽  
Eerappa Rajakumara
Keyword(s):  
Catalytic Activity ◽  
Repair Process ◽  
Single Strand ◽  
Genomic Integrity ◽  
Post Translational Modification ◽  
Dna Breaks ◽  
Binding Studies ◽  
Competition Binding ◽  
Multiple Reader ◽  
Stimulation Of

Poly(ADP-ribosyl)ation is a post translational modification, predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single strand (SSB) and double strand (DSB) DNA breaks are bonafide stimulators of PARP1 activity. We identified that, in addition, single strand (ss) DNA also binds and stimulates the PARP1 activity. Poly(ADP-ribose) (PAR) is chemically similar to ssDNA. However, PAR mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT and WGR, hitherto uncharacterized, as PAR-specific reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared to PAR, but do not bind to DNA. Conversely, N-terminal domains, ZnF1 and ZnF2, of PARP1 recognize DNA but not PAR. Further competition binding studies suggest that PAR binding, allosterically releases DNA from PARP1. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampers the DNA dependent stimulation. Altogether, our work discovers dedicated PAR and DNA reader domains of the PARP1, and uncovers a novel mechanism of allosteric stimulation of the catalytic activity of PARP1 but retardation of DNA-dependent activities of PARP1 by its catalytic product PAR.

Muscarinic receptors in MDCK cells are coupled to multiple messenger systems

1992 ◽  
Vol 263 (6) ◽  
pp. C1289-C1294 ◽  
Author(s):  
D. Mohuczy-Dominiak ◽  
L. C. Garg
Keyword(s):  
Adenylate Cyclase ◽  
Muscarinic Receptors ◽  
Pertussis Toxin ◽  
Radioligand Binding ◽  
Mdck Cells ◽  
Adenylate Cyclase System ◽  
Dependent Manner ◽  
Muscarinic Antagonists ◽  
Binding Studies ◽  
Camp Formation

Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated that high-affinity specific muscarinic receptors coupled to the phosphoinositide system are present in these cells. To determine whether muscarinic receptors in MDCK cells are linked negatively to the adenylate cyclase system, we measured the effect of muscarinic agonists and antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at 10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1, respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and forskolin were blocked by arecoline, a cholinergic agonist, in a concentration-dependent manner. The arecoline response was blocked by treatment of the cells with pertussis toxin. The inhibition by arecoline of forskolin-stimulated cAMP formation was reversed by various muscarinic antagonists in the following order of potency: 4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol > pirenzepine > methoctramine. This order of potency of muscarinic antagonists is similar to that observed in our radioligand binding studies and is consistent with the M3 subtype of muscarinic receptors. Our results indicate that muscarinic receptors in MDCK cells are coupled negatively to the adenylate cyclase system via pertussis toxin-sensitive G protein. It is concluded that this intracellular system may at least be partially responsible for the action of cholinergic agonists in these cells and in the kidney.

EGTA-sensitive and -insensitive forms of particulate adenylate cyclase in rat cerebral cortex: regulation by divalent cations and GTP

1985 ◽  
Vol 63 (8) ◽  
pp. 1007-1016 ◽  
Author(s):  
P. V. Sulakhe
Keyword(s):  
Enzyme Activity ◽  
Cerebral Cortex ◽  
Adenylate Cyclase ◽  
Gray Matter ◽  
Rank Order ◽  
Divalent Cations ◽  
Rat Cerebral Cortex ◽  
Chloride Salt ◽  
Low Concentrations ◽  
Stimulation Of

Interactions of several divalent cations (Mn2+, Ca2+, Co2+, Sr2+, and Zn2+) with EGTA-inhibitable adenylate cyclase were investigated in washed membranes (particles) isolated from the gray matter of rat cerebral cortex. The EGTA-inhibitable (called sensitive) enzyme activity was assayed in the presence of Triton X-100 since this detergent caused a marked increase (up to 20-fold) in the enzyme activity. The effects of various divalent metals (all added as chloride salt) indicated the presence of two distinct sites called site I and site II. At low concentrations (less than micromolar) Mn2+, Co2+, and Ca2+ increased (up to 10-fold) the enzyme activity to the same extent and appeared to act via binding to site I (high affinity site). The rank order of affinity was Mn2+ ≥ Co2+ > Ca2+. Zn2+ showed the highest affinity and Sr2+ the lowest towards binding to site I; both these metals increased the enzyme activity to lesser extents than Mn2+, Co2+, or Ca2+. GTP was not required for the stimulation of this enzyme by low concentrations of Ca2+. The interaction of Mn2+ with site II (low affinity site) caused further increase in the enzyme activity, whereas Co2+, Ca2+, and Sr2+ were inhibitory at concentrations >10 μM. Isolated fraction contained loosely and tightly associated pools of calmodulin. Myelin basic protein, but not calcineurin, inhibited the EGTA-sensitive adenylate cyclase activity. The EGTA-insensitive enzyme activity was increased by norepinephrine by mechanisms that depended on GTP and was inhibited by Ca2+. The stimulation of the EGTA-insensitive enzyme modulated the Mg2+ requirement such that Mg2+ binding to the low affinity site (site II) apparently occurred with higher affinity. The likely significance of these results is discussed with regard to (i) the presence of two classes of adenylate cyclase in rat cerebral cortex gray matter and (ii) the regulation of their activities by calmodulin-requiring and GTP-requiring mechanisms.

scholarly journals Gene and cDNA cloning and characterization of the mouse V3/V1b pituitary vasopressin receptor

1999 ◽  
Vol 22 (3) ◽  
pp. 251-260 ◽  
Author(s):  
MA Ventura ◽  
P Rene ◽  
Y de Keyzer ◽  
X Bertagna ◽  
E Clauser
Keyword(s):  
Inositol Phosphate ◽  
Rank Order ◽  
Single Gene ◽  
Dependent Manner ◽  
Binding Studies ◽  
V1b Receptor ◽  
Competition Binding ◽  
Receptor Cdna ◽  
Dose Dependent

The gene of the mouse V3/V1b receptor was identified by homology cloning. One of the genomic clones contained the entire coding sequence. The cDNA presented high identity with rat (92%) and human (84%) sequences. Southern blot analysis indicated the existence of a single gene. Tissue distribution was studied by RT-PCR. The major site of expression was the pituitary. A faint signal was also present in hypothalamus, brain, adrenal, pancreas and colon. The mouse corticotroph cell line, AtT20, did not express the transcript. In order to confirm the identity of the sequence, the V3/V1b receptor cDNA was cloned and stably expressed in CHO-AA8 Tet-Off cells under the control of tetracycline. When transfected cells were treated with arginine vasopressin (AVP), inositol phosphate production increased in a dose-dependent manner, indicating that the V3/V1b receptor couples to phospholipase C. Moreover, AVP did not stimulate cAMP production. Binding studies with [3H]AVP indicated that the affinity of the mouse V3/V1b receptor (Kd=0.5 nM) is similar to that reported for rat and human receptors. The rank order of potency established in competition binding experiments with different analogues was representative of a V3/V1b profile, distinct from V1a and V2. However, significant differences were found between human and mouse receptors tested in parallel. Thus the pharmacology of V3/V1b receptors can not be transposed among different species.

Receptor-G-protein signalling in Alzheimer's disease

2001 ◽  
Vol 67 ◽  
pp. 163-175 ◽  
Author(s):  
Richard F. Cowburn ◽  
Cora O'Neill ◽  
Willy L. Bonkale ◽  
Thomas G. Ohm ◽  
Johan Fastbom
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Protein Kinase ◽  
G Protein ◽  
Radioligand Binding ◽  
Phosphoinositide Hydrolysis ◽  
Binding Studies ◽  
Neurofibrillary Changes ◽  
Hydrolysis Pathway ◽  
Stimulation Of

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.

The characteristics of β-adrenergic binding sites on pancreatic islets of Langerhans

1986 ◽  
Vol 111 (2) ◽  
pp. 263-270 ◽  
Author(s):  
J. M. Fyles ◽  
M. A. Cawthorne ◽  
S. L. Howell
Keyword(s):  
Insulin Secretion ◽  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Islets Of Langerhans ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Islet Cells ◽  
Pancreatic Islets Of Langerhans ◽  
Β Adrenergic Receptors ◽  
Stimulation Of

ABSTRACT The sympathetic nervous system is believed to play a part in the control of insulin release from the pancreatic islets of Langerhans. Stimulation of α-adrenoceptors is thought to inhibit the release of insulin whereas stimulation of β-adrenoceptors enhances insulin release. The present experiments were conducted to establish the existence of β-adrenergic receptors on guinea-pig and rat islet cells and to quantify them using the selective β-adrenergic ligands [3H]dihydroalprenolol (DHA) and [125I]cyanoiodopindolol (CYP). Guinea-pig islets had 62 fmol β-adrenoceptors/mg protein using [3H]DHA, corresponding to 43 700 binding sites/cell and 25 fmol β-adrenoceptors/mg protein using [125I]CYP, corresponding to 17 400 sites/cell. Rat islet cells were found to have 4·6 fmol β-adrenoceptors/mg protein using [125I]CYP, corresponding to 7200 sites/cell. Adenylate cyclase activation exhibited a positive dose–response relationship when exposed to the β-adrenoceptor agonist isoprenaline, with a maximum response (190 ± 21% above basal) at 10 μmol isoprenaline/l. This response was abolished with 1 μmol/l of the β-adrenergic antagonist 1-alprenolol. Insulin secretion in the presence of 10 mmol glucose/l, but in the absence of the α-adrenoceptor blocker phentolamine, was not affected by 10 μmol isoprenaline/l. However, perifusion experiments showed that secretion of insulin from isolated rat islets in the presence of 10 mmol glucose/l was significantly increased (332%) by 10 μmol isoprenaline/l in the presence of 10 μmol phentolamine/l. These results suggest that binding of selective radio-labelled ligands occurs to β-adrenergic receptors on the B cell surface of the islets of Langerhans, and that these receptors are functionally coupled to insulin secretion through modulation of adenylate cyclase activity. J. Endocr. (1986) 111, 263–270

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