Biomedical subjects

C Nanoff

Publications and source records attributed to C Nanoff.

At least 37 records · Page 2Linked to original sources

PubMed1997

Coupling cofactor--a novel regulatory component in G protein-mediated signalling?

The model for studying the mechanism of G protein-mediated signalling cannot account for the observation that high-affinity binding of agonists to many different receptors is not dissociated by the addition of high concentrations of guanine nucleotides. Using the cerebral A1-adenosine receptor as a model system, we have recently identified a component which is responsible for this phenomenon. This protein, termed the coupling cofactor, can be solubilized from brain membranes and chromatographically resolved from both the G proteins and the receptor. Following reconstitution into appropriate acceptor membranes, the coupling cofactor confers resistance of high-affinity agonist binding to guanine nucleotides. The coupling cofactor acts as a brake and limits receptor-dependent signal amplification; in addition, it is a candidate for participating in the higher level organization of receptors and G proteins in membranes and in the membrane-delimited cross-talk between individual receptors. Here, we present a working hypothesis on the possible biological roles of the coupling cofactor.

Animals↗

PubMed1996

Thiophosphorylation of the G protein beta subunit in human platelet membranes: evidence against a direct phosphate transfer reaction to G alpha subunits.

A direct phosphate transfer reaction from the G protein beta subunits to either Gs alpha or Gi alpha has been proposed to account for the ability of thiophosphorylated transducin beta gamma-dimers to bidirectionally regulate adenylyl cyclase activity in human platelet membranes. We searched for experimental evidence for this reaction. Incubation of human platelet membranes with [35S]guanosine-5'-(3-O-thio)triphosphate ([35S]GTP gamma S) results in the predominant incorporation of [35S]thiophosphate into a 36-kDa protein, which comigrates with the G protein beta subunit and is immunoprecipitated by a beta subunit-specific antiserum. Thiophosphorylation of the beta subunit is specific for guanine nucleotides and abolished by the histidine-modifying agent diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of [35S]thiophosphorylated beta subunits is accelerated in the presence of GDP, but not ADP, UDP, or guanosine-5'-(2-O-thio)diphosphate. Neither the thiophosphorylation nor the dephosphorylation is sensitive to receptor agonists (alpha 2-adrenergic, A2 adenosine, thrombin, or insulin), and purified G protein alpha subunits do not act as thiophosphate donors. An approach was designed to demonstrate direct thiophosphate transfer to protein-bound nucleotides; platelet membranes were sequentially exposed to NaIO4, NaCNBH3, and NaBH4, an oxidation-reduction step that covalently incorporates prebound nucleotides into proteins. Under these conditions, multiple radiolabeled proteins are visualized on subsequent addition of [35S]GTP gamma S. This reaction is specific because both oxidation and reduction are required and pretreatment of platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyrocarbonate blocks the subsequent labeling in oxidized and reduced membranes. The G protein beta subunit may participate in this thiophosphate transfer reaction. Most important, however, no labeled G protein alpha subunits (Gs alpha and Gi alpha) were recovered by immunoprecipitation from oxidized and reduced membranes subsequent to the addition of [35S]GTP gamma S. Thus, our results clearly rule out the existence of a postulated G protein activation by phosphate transfer reactions, which lead to the formation of GTP from GDP prebound to the alpha subunit.

Adenosine Diphosphate↗

PubMed1996

Suramin analogues as subtype-selective G protein inhibitors.

G protein alpha subunits expose specific binding sites that allow for the sequential, conformation-dependent binding of protein reaction partners, e.g., G protein beta gamma dimers, receptors, and effectors. These domains represent potential sites for binding of low-molecular-weight inhibitors. We tested the following suramin analogues as G protein antagonists: 8-(3-nitrobenzamido)-1,3,5-naphtalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)benzamido)-1,3,5-naphtalenetrisulfonic++ + acid NF018), 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphtalenetri sulfonic acid) (NF023), 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1-phe nylene))bis-(1,3, 5-naphtalenetrisulfonic acid) (NF037), and suramin. The compounds suppressed [35S]GTPgammaS binding to purified, recombinant G protein alpha subunits, an effect that is due to inhibition of GDP release. Suramin is selective for recombinant Gsalpha-s (EC50 values o f approximately 240 nM; rank order of potency, suramin > NF037 > NF023 > NF018 > NF007), whereas NF023 is selective for recombinant Gi alpha-1 and recombinant Go alpha (EC50 value of approximately 300 nM; rank order of potency, NF023 > / = NF037 > suramin >0 NF018 > NF007). Selectivity was also demonstrated on a cellular level. In rat sympathetic neurons, alpha-2-adrenergic and muscarinic receptor-dependent inhibition of the voltage-sensitive calcium current is mediated by Gi/Go, whereas inhibition by vasoactive intestinal peptide (VIP) is mediated by Gs. Calcium current inhibition by alpha2-adrenergic and muscarinic receptors was greatly reduced when 100 microM NF023 was applied intracellularly, whereas the response to VIP was unaffected; in contrast, the response to VIP was blunted only with 100 microM suramin in the recording pipette. The suramin analogues do not interfere with the interaction between alpha subunits and G protein beta gamma dimer but compete with binding of the effector. The addition of purified adenylyl cyclase reverses the inhibitory effect of suramin on the rate of [35S]GTPgammaS binding to recombinant Gsalpha-s, indicating direct competition for a common site; similarly, immunoprecipitation by an antibody directed against an epitope of the effector binding site is inhibited by suramin. Our results show that it is possible to design G protein inhibitors that target the effector binding site on the alpha subunits.

Animals↗

PubMed1996

Inhibition of receptor/G protein coupling by suramin analogues.

Suramin analogues act as direct antagonists of heterotrimeric G proteins because they block the rate-limiting step of G protein activation (i.e., the dissociation of GDP prebound to the G protein alpha subunit). We have used the human brain A1 adenosine receptor and the rat striatal D2 dopamine receptor, two prototypical Gi/G(o)-coupled receptors, as a model system to test whether the following analogues suppress the receptor-dependent activation of G proteins: 8-(3-nitrobenzamido)-1,3,5-naphthalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)-benzamido)-1,3,5-naphthalenetrisulfonic acid (NF018); 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalenetr isulfonic acid) (NF023); 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1- phenylene)) bis(1,3,5-naphthalenetrisulfonic acid) (NF037); and suramin. Suramin and its analogues inhibit the formation of the agonist-specific ternary complex (agonist/receptor/G protein). This inhibition is (i) quasicompetitive with respect to agonist binding in that it can be overcome by increasing receptor occupancy but (ii) does not result from an interaction of the analogues with the ligand binding pocket of the receptors because the binding of antagonists or of agonists in the absence of functional receptor/G protein interaction is not affected. In addition to suppressing the spontaneous release of GDP from defined G protein alpha subunits, suramin and its analogues reduce receptor-catalyzed guanine nucleotide exchange. The site, to which suramin analogues bind, overlaps with the docking site for the receptor on the G protein alpha subunit. The structure-activity relationships for inhibition of agonist binding to the A1 adenosine receptor (suramin > NF037 > NF023) and of agonist binding to the inhibition D2 dopamine receptor (suramin = NF037 > NF023 > NF018) differ. Thus, NF037 discriminates between the ternary complexes formed by the agonist-liganded D2 dopamine receptors and those formed by the A1 adenosine receptor with > 10-fold selectivity. Therefore, our results also show that inhibitors can be identified that selectively uncouple specific receptor/G protein tandems.

Animals↗

PubMed1995

Species differences in A1 adenosine receptor/G protein coupling: identification of a membrane protein that stabilizes the association of the receptor/G protein complex.

Reconstitution experiments with purified components reproduce the basic characteristics of receptor/G protein coupling, i.e., GTP-sensitive high affinity agonist binding and receptor-promoted GTP binding. However, the interaction of agonists with the A1 adenosine receptor in rat and bovine but not human brain membranes deviates from the ternary complex model since the agonist/receptor/G protein complex cannot be dissociated by high concentrations (> or = 100 microM) of the hydrolysis-resistant analogue GTP gamma S. The reason for this phenomenon referred to as a "tight coupling mode" has remained enigmatic. We show that it is attributable to a distinct membrane protein, which we labeled the coupling cofactor. Extraction of the protein from rat brain membranes with the detergent 3[3-(cholamidopropyl)diamethylammonio]-1-propanamium increased the potency of GTP gamma S by 1000-fold. After extraction, the potency was comparable to that in human brain membrane. Detergent extracts from rat brain membranes were used to resolve the component from solubilized receptors and G protein alpha and beta gamma subunits by sequential DEAE-Sephacel chromatography and Superose gel filtration (molecular weight of approximately 150 kDa in 3[3-(cholamidopropyl)diamethylammonio]-1-propanamium). Coupling cofactor restored guanine nucleotide refractoriness in a concentration-dependent manner to both detergent-extracted rat brain membranes and, albeit with lower affinity, human brain membranes. However, in human brain extracts, cofactor activity was detectable on reconstitution with rat acceptor membranes, indicating an intrinsic difference between rat and human receptors in their ability to interact with the cofactor. With high amounts of coupling cofactor present, GTP gamma S no longer decreased but rather increased agonist affinity. Readdition of partially purified coupling cofactor to acceptor membranes reduced the rate of A1 adenosine receptor-mediated G protein turnover. These observations show that the component identified traps the ternary agonist/receptor/G protein complex in a stable conformation, impedes signaling of the A1 adenosine receptor, and thereby regulates the level of signal amplification.

Animals↗

PubMed1994

Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor.

The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G protein specificity suggesting that species homologues of receptors may use different signaling mechanisms.

Animals↗

PubMed1994

2',3'-Dialdehyde GTP as an irreversible G protein antagonist. Disruption and reconstitution of G protein-mediated signal transduction in cells and cell membranes.

The 2',3'-dialdehyde analogue of GTP, oGTP, was devised as an irreversible antagonist of regulatory GTP-binding proteins (G proteins). Here, we show that oGTP uncouples transmembrane signaling mediated by a set of distinct G proteins both in isolated membranes and in whole cells. In human platelet membranes, pretreatment with oGTP suppressed receptor- and G protein-controlled regulation of adenylyl cyclase activity. In chick neuronal cells, inhibition of the voltage-sensitive Ca(2+)-current by various membrane receptors (alpha 2-adrenergic, somatostatin, GABAB) was eliminated when oGTP was applied intracellularly in the whole cell patch-clamp configuration. Disruption of endogenous signaling pathways by oGTP occurred through specific blockage of the GTP-binding site of G protein alpha-subunits by the following criteria: (i) pretreatment of membranes with oGTP blocked direct G protein activation by guanine nucleotides as well as labeling of Gs alpha and Gi alpha with the photoaffinity probe [alpha-32P]GTP azidoanilide. (ii) The effect of oGTP was antagonized by the simultaneous introduction of guanosine 5'-(3-O-thio)triphosphate into the patch-clamped cell. (iii) The time to onset of action was similar for oGTP and guanosine 5'-O-thio)diphosphate. (iv) Inactivation of G protein-dependent signaling was overcome by substituting G protein alpha-subunits. Addition of both the short and long form of recombinant Gs alpha (rGs alpha-s and rGs alpha-L) restored guanine nucleotide-dependent adenylyl cyclase activity to oGTP-treated platelet membranes with rGs alpha-L being approximately 3-10-fold more potent than rGs alpha-s. This apparent preference was due to the intrinsically different activation rates of rGs alpha-L and rGs alpha-s. When reconstituted with exogenous rGs alpha, the A2-adenosine receptor did not discriminate among the two forms of rGs alpha. Thus, Gs alpha-L is the primary determinant of basal cAMP formation in platelets. In contrast, neither the addition of various recombinant subtypes of Gi/o nor purified bovine brain beta gamma-dimers reconstituted adenylyl cyclase inhibition in oGTP-treated membranes. All subtypes of Gi alpha stimulated adenylyl cyclase. In the presence of rGs alpha, a conditional stimulation by beta gamma-dimers was observed. This pattern of stimulation shows that platelet adenylyl cyclase is a type II-like isoform. Either a differently modified G protein or an ancillary GTP-binding component is required for adenylyl cyclase inhibition in platelets. oGTP can be considered a useful tool for disruption and reconstitution of transmembrane signaling mediated by presumably all classes of heterotrimeric G proteins.

Adenylyl Cyclases↗

PubMed1994

Structural and functional characterization of the interaction between 2',3'-dialdehyde guanine nucleotide analogues and the stimulatory G protein alpha-subunit.

We have searched for irreversible ligands which target the guanine nucleotide binding pocket of G protein alpha-subunits by testing the ability of periodate-oxidized 2',3'-dialdehyde guanine nucleotide analogues of GTP (oGTP) and GTP gamma S (oGTP gamma S) to bind to the recombinant alpha-subunit of the stimulatory G protein, rGs alpha-s. oGTP and oGTP gamma S bind to rGs alpha-s in a quasi-irreversible manner via formation of a Schiff's base, which can be reduced with borhydrid resulting in covalent incorporation of [alpha-32P]oGTP and [35S]oGTP gamma S into rGs alpha-s. When bound to rGs alpha-s, oGTP is hydrolyzed and traps the protein in the inactive conformation, while oGTP gamma S persistently activates rGs alpha. Thus, oGTP and oGTP gamma S act as irreversible G protein antagonist and agonist, respectively, and represent a pair of nucleotide analogues suitable as functional and structural tools. Cleavage of covalently labeled rGs alpha-s with cyanogen bromide generates several labeled fragments. Labeled fragments were assigned to the G1 and G4 region of the guanine nucleotide binding pocket using sequence-specific antisera. An additional, labeled fragment was identified by amino-terminal sequencing and corresponded to the helix alpha A in the recently determined crystal structure of the transducin alpha-subunit (Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663). In the oGDP-liganded conformation, incorporation occurs predominantly into the G1-fragment, while [35S]oGTP gamma S labels the additional fragments to a similar extent indicating tight packing around the guanine nucleotide binding pocket in the active conformation. Furthermore, rGs alpha-s contains a single acid cleavable bond (Asp317-Pro318), such that formic acid releases a carboxyl-terminal fragment from [alpha-32P]oGTP- and [35S]oGTP gamma S-liganded rGs alpha-s. This fragment contains a single lysine residue (Lys324) which is only labeled by [35S]oGTP gamma S. Lys324 is unique to Gs alpha and lies within its effector binding region. Hence, during the switch from the inactive to the active state, this region undergoes a major conformational change that moves it closer to the nucleotide binding pocket.

Adenylyl Cyclases↗

PubMed1993

Solubilization and characterization of the A2-adenosine receptor.

Binding of [3H]CGS 21680, an agonist radioligand selective for A2-adenosine receptors (A2AR), to membranes and solubilized preparations from bovine brain striatum revealed labelling of a single high affinity binding state. In membranes, guanine nucleotides per se were ineffective in modulating agonist binding whereas cations, Na+ and Mg++, had distinct effects. The addition of NaCl (200 mM) as well as the Mg(++)-free preparation of membranes led to a significant decrease in binding affinity and the number of binding sites. Moreover, the presence of Na+ was required for the demonstration of a guanine nucleotide effect, i.e. a decrease in maximal binding. Following solubilization, agonist-A2AR interactions were sensitive to guanine nucleotides even in the absence of Na+; guanine nucleotides and Na+ had additive effects in reducing the number of binding sites. Moreover, the effect of GTP was reversible, i.e. binding returned to control levels upon removal of the nucleotide. This suggests the A2AR and its G protein (presumably Gs) are solubilized as a functional unit and may not dissociate even in the presence of GTP following solubilization. We, therefore, believe that a "tight" association exists between receptor and G protein (Gs), and that guanine nucleotides and sodium act at different sites on the R-G complex. Drawing an analogy with similar observations on the avian beta-adrenergic receptor (Hertel et al, J. Biol. Chem. 265:17988-94, 1990; Parker & Ross, J. Biol. Chem. 266:9987-96, 1991) we postulate that the regulatory features of the A2AR can be attributed to a distinct receptor domain that interacts with cellular regulatory elements.

Adenosine↗

PubMed1992

Sensitization of dopamine-stimulated adenylyl cyclase in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys and patients with idiopathic Parkinson's disease.

Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys and humans with idiopathic Parkinson's disease and compared with the activity in control tissue. No differences between parkinsonian and control tissue were found in the presence of 20 mM NaCl. However, when 120 mM NaCl was included in the assay medium, a significantly higher increase in the Vmax of dopamine-stimulated adenylyl cyclase activity was observed in the caudate of MPTP-parkinsonian rhesus monkeys and the putamen of patients with idiopathic Parkinson's disease. No such sensitization was seen in the MPTP-treated rhesus putamen or human Parkinson's disease caudate tissue. A role of D2 receptors in this sensitization could be ruled out by the concomitant use of the D2 antagonist l-sulpiride and by [3H]spiperone saturation analysis of the D2 receptor density, which was found at control level in the caudate tissue of MPTP-treated rhesus monkeys. Similarly, on the basis of saturation binding with the D1 selective ligand 125I-SCH 23982, there was no difference in caudate nucleus D1 receptor densities between control and MPTP-treated monkeys. Our results point to a region-specific functional sensitization of D1 receptors as a consequence of severe dopaminergic denervation of the striatum and suggest the possibility of a therapeutic potential of a D1 agonist with full intrinsic activity in Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1991

Characterization of the beta-adrenoceptor blocking property of diprafenone in rats: stereoselective interaction, subtype specificity, and sensitization.

Because of their structural relationship to propranolol, propafenone and diprafenone display beta-adrenoceptor blocking activity in addition to their class Ic antiarrhythmic property. As demonstrated in membranes derived from rat ventricle (predominantly beta 1-adrenoceptors) and rat lung tissue (predominantly beta 2-adrenoceptors), the (-)-enantiomer of diprafenone was about four times more potent (Ki 6.6 nmol/L) than the (+)-enantiomer in displacing [125I]iodocyanopindolol (ICYP) binding. The Ki values for the (+)- and (-)-stereoisomer, racemic (+/-)-diprafenone, and 5-hydroxydiprafenone, the main metabolite of diprafenone in humans, were approximately 2.5 times lower in lung than in ventricular membranes, suggesting very low beta 2-selectivity for diprafenone. The regulatory effect of diprafenone on ventricular beta-adrenoceptors was studied in rats in vivo by prolonged i.p. administration of the drug. Density of beta-adrenoceptors was estimated by ICYP saturation binding after 2-day (4 or 20 mg/kg, b.i.d.) and after 7-day treatment (4 mg/kg, b.i.d.), respectively. For control purposes, different groups of animals were treated with propranolol (1.7 mg/kg, b.i.d., i.p.), isoprenaline (0.1 mg/kg/h via subcutaneously implanted osmotic minipumps), and vehicle (0.9% NaCl) only. Whereas propranolol and isoprenaline produced an increase (7-day treatment) and a decrease (2- and 7-day treatment) in beta-adrenoceptors, respectively, diprafenone did not produce any change in beta-adrenoceptor number, irrespective of the dose and duration of treatment used. Furthermore, the combined administration of diprafenone and isoprenaline did not antagonize isoprenaline-induced down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗

PubMed1991

The A2 adenosine receptor: guanine nucleotide modulation of agonist binding is enhanced by proteolysis.

Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125I-2-[4-(2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminnocarbonyl]ethyl)phenyl]ethylamino-5'-N- ethylcarboxamidoadenosine (125I-PAPA-APEC) and its photoaffinity analog 125I-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is labeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125I-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr approximately 47,000 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory GS-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit striatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5'-(beta,gamma-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by only 28% in the Mr 47,000 receptor protein. Our data, therefore, suggest that, unless proteolysis occurs, the A2AR in all tissues studied is tightly associated with the GS protein and displays minimal guanine nucleotide modulation of agonist binding, which makes the A2AR an atypical stimulatory receptor.

Adenosine↗

PubMed1990

[Role of G protein-mediated signal transduction in molecular pharmacodynamics].

Hormones, neurotransmitter and autacoid receptors, localized on the plasma membrane, do not interact directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger), but control their target systems via activation of an intermediary guanine nucleotide binding protein on G protein, which serves as signal transducer. Traffic of these pathways is regulated via a GTP (on)-GDP (off) switch, which is triggered by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. Receptor desensitization is a main criterion of G protein-coupled receptors with important pharmacological implications. Multiple mechanisms are responsible for the loss of sensitivity that follows against exposure. The process is initiated by uncoupling the receptor from its G protein, which is due to receptor phosphorylation by specific kinases. In the case of the beta-adrenergic receptor, two particular kinases - beta-adrenergic receptor kinase (beta ARK) and protein kinase A--are involved. Further steps of desensitization are receptor sequestration or internalization, an event as rapid and transient as receptor uncoupling, and receptor downregulation, which requires more prolonged agonist exposure. Finally, antagonists are able to induce a receptor-G protein interaction in a reverse manner to agonists. Whereas agonists stimulate both, the GDP dissociation from the G protein and the association of GTP, antagonists markedly decrease GTP association. Moreover, in the turkey erythrocyte adenylyl cyclase system antagonists decrease the GTP-stimulated adenylyl cyclase activity almost at basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1990

P2-, but not P1-purinoceptors mediate formation of 1, 4, 5-inositol trisphosphate and its metabolites via a pertussis toxin-insensitive pathway in the rat renal cortex.

1. The adenosine receptor (P1-purinoceptor) agonists N6-cyclopentyladenosine and N-5'-ethyl-carboxamidoadenosine at concentrations up to 10 mumols 1(-1) affected neither basal, nor noradrenaline- and angiotensin II-stimulated formation of inositol-1-phosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate in slices of rat renal cortex. 2. In contrast, adenine nucleotides (P2-purinoceptor agonists) markedly stimulated inositol phosphate formation. The observed rank order of potency adenosine-5'-O-(2-thiodiphosphate) (EC50 39 mumols 1(-1] greater than adenosine-5'-O-(3-thiotriphosphate) (587) greater than or equal to 5'-adenylylimidodiphosphate (App(NH)p, 899) greater than adenylyl-(beta, gamma-methylene)-diphosphate (4,181) was consistent with the interaction of the compounds with the P2Y-subtype of P2-purinoceptors. AMP and the ADP analogue (alpha, beta-methylene)-adenosine-5'-diphosphate were ineffective. ATP and ADP (less than or equal to 10 mmol 1(-1] did not produce a consistent increase, owing to their hydrolytic degradation in the incubation medium. 3. Whereas the inositol phosphate response to App(NH)p was linear only up to 5 min incubation, the time-dependent stimulation of noradrenaline declined at a slower rate. Following pre-exposure of the renal cortical slices to App(NH)p, renewed addition of App(NH)p caused no further enhancement in the accumulation of inositol phosphates, whilst noradrenaline was still capable of eliciting a response. This suggests that the apparent loss of responsiveness to App(NH)p is not due to substrate depletion or enzymatic inactivation, but most likely attributable to homologous desensitization of the purinoceptor. 4. Pretreatment of the animals with pertussis toxin caused a substantial reduction of functional Gi-protein, as indicated by the lack of [32P]-NAD incorporation in a membrane preparation of the renal cortex. Nevertheless, the increase in inositol phosphate formation induced by noradrenaline, angiotensin II, and App(NH)p was not significantly impaired. 5. We conclude that P2 gamma-purinoceptors are present in the renal cortex; these receptors stimulate formation of inositol phosphates via a pertussis toxin-insensitive pathway and undergo homologous desensitization. On the other hand, our results suggest that renal A,-adenosine receptors do not use stimulation of phosphoinositide breakdown as a transmembrane signalling system.

Adenine Nucleotides↗

PubMed1989

Differences in the GTP-regulation of membrane-bound and solubilized A1-adenosine receptors.

Using [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a 3H-labeled A1-selective adenosine antagonist with high affinity and extremely low non-specific binding, it was possible to quantitatively evaluate the effect of GTP on agonist binding. Competition experiments on [3H]DPCPX binding to guinea-pig cerebral cortical membranes in the absence of GTP showed a high- and a low-affinity state for adenosine receptor agonists (82/18% for N6-cyclopentyl-adenosine). Addition of 1 mmol/l GTP only partially converted the high-affinity state of the A1-adenosine receptor into a low-affinity state. This failure of complete conversion from high- to low-affinity state was also seen in membranes from rat testes under the same experimental conditions and, moreover, in guinea-pig brain membranes under different experimental conditions, such as in the presence of Na+ or when free Mg2+ has been reduced by EDTA. The only difference was that in the absence of Mg2+ the high-affinity state of the A1-receptor was markedly smaller than in the presence of Mg2+ (36% vs. 82%). By contrast, in the solubilized state of the receptor total conversion of all receptors into the low-affinity state was obtained upon addition of 1 mmol/l GTP. Reduction of binding of the agonist radioligand [125]iodo-N6-(4-hydroxyphenylisopropyl)-adenosine with increasing concentrations of GTP and Gpp(NH)p demonstrated that the guanine nucleotide affinity to the solubilized A1-receptor was more than 100-fold higher than to the membrane-bound receptor. Hence, the incomplete transition of the high-affinity into the low-affinity state of the membrane-bound A1-receptor upon addition of GTP may be attributable to the low affinity of the membrane-bound receptor-G-protein complex for GTP.

Adenylyl Cyclases↗

PubMed1989

A different desensitization pattern of cardiac beta-adrenoceptor subtypes by prolonged in vivo infusion of isoprenaline.

(-)Isoprenaline was continuously administered to rats at a rate of 0.4 mg/kg/h for 7 days via subcutaneously (s.c.) implanted osmotic minipumps. This treatment induced cardiac hypertrophy and a marked decrease in basal as well as catecholamine-stimulated adenylate cyclase activity in a ventricular plasma membrane fraction. The total number of beta-adrenoceptors was downregulated by one-half the amount of the receptor sites obtained in a control group. However, in the isoprenaline-treated group, the beta 2-adrenoceptors constituted a significantly smaller proportion of the total beta-adrenoceptor population (28%) than in the control group (50%). Transformation of these relative into absolute values indicates that prolonged isoprenaline treatment induced a significantly higher downregulation of beta 2- than of beta 1-adrenoceptors. The fact of a different beta-adrenoceptor desensitization pattern in response to in vivo administration of nonselective beta-adrenergic agonists therefore must be taken into consideration when desensitization is used as a method for determination of subtype selectivity of an agonist per se. However, we were unable to detect the "lost" beta-adrenoceptors in a light vesicular fraction. In our study, this fraction was not separable from plasma membranes, as substantiated by levels of plasma membrane markers as high as in the plasma membrane fraction and by a guanine nucleotide-dependent adenylate cyclase activity.

Adenylyl Cyclases↗

PubMed1989

[Follow-up study of severely overweight adolescents 4 years following inpatient weight loss with a low calorie protein-carbohydrate diet].

Very low calorie protein/carbohydrate diets have proved to be efficient and safe in the treatment of obesity in childhood and early adolescence. The follow-up study at issue shows that subsequent to short-term therapy no further reduction in weight is to be expected without adequate care following the diet. Between 1980 and 1983, 27 adolescent patients were treated for severe obesity (78.12% +/- 18.3% overweight, mean +/- s.e.m.) at the metabolic ward of the department of pediatrics, University of Vienna. The administration of a very low calorie protein/carbohydrate diet (VLCD) for 26 (+/- 7) days led to a considerable reduction in overweight (8.1 +/- 2.9 kg). On dismission the percentual overweight had decreased to 59.4 (+/- 24)%. After 4.4 (+/- 0.7) years the weight of 11 of the 27 patients could be controlled, 10 patients refused further cooperation and the remaining 6 could not be addressed. The body mass index (weight/height) had risen from 29.4 (+/- 1.2) to 43.0 (+/- 6.0) with 10 of 11 patients, corresponding to a percentual overweight of 88.2 (+/- 18.1)% at the follow-up date. Only one girl had reduced overweight by 45%, the BMI had fallen from 33.9 to 27.1. Furthermore, our data suggest that obesity induces a significant increase in blood pressure in adolescents. This rise is apparently independent of the age related increase in blood pressure and revertible by weight loss. The results we have to hand show that long-term weight reduction has only been achieved in 1 of 11 cases, with none of the adolescents having participated regularly in an outpatient after-care program.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1987

Stimulation of adenylate cyclase activity via A2-adenosine receptors in isolated tubules of the rabbit renal cortex.

Adenylate cyclase activity in a tubular fraction obtained from rabbit renal cortex was stimulated by typical adenosine receptor agonists with a rank order of potency NECA (5'-(N-ethyl-carboxamido)-adenosine) (EC50 = 0.48 mumol/l) greater than R-PIA [(-)N6 (R-phenylisopropyl)-adenosine] (3.22 mumol/l). The stimulatory effect of NECA was competitively antagonized by 8-phenyltheophylline. Contamination of the tubular fraction with glomeruli and microvessels was less than 2%, as verified by tissue renin determination and could, therefore, be ruled out as being responsible for the observed effect. Tubular A2-adenosine receptors are probably involved in the control of renal electrolyte secretion and may represent the site of action of methylxanthines.

Adenosine↗