Biomedical subjects

J Linden

Publications and source records attributed to J Linden.

At least 91 records · Page 5Linked to original sources

PubMed1994

Effects of xanthine amine congener on hypoxic coronary resistance and venous and epicardial adenosine concentrations.

OBJECTIVE: The aim was to define the contributions of interstitial and vascular adenosine in regulating coronary vascular resistance during hypoxia. To help in the assessment of adenosine in the vasodilator response, a potent adenosine receptor antagonist, xanthine amine congener (XAC), was used to block adenosine receptors. METHODS: Seven isolated guinea pig hearts were perfused at constant flow with Krebs buffer. Coronary vascular resistance was determined during normoxia (95% O2) and mild hypoxia (60% O2) in the absence or presence of 200 or 400 nM XAC. Interstitial fluid was sampled by the epicardial disc technique and the interstitial concentration of XAC (ISF[XAC]) was determined directly by a radioreceptor assay or as tritiated XAC. Venous and epicardial concentrations of adenosine were determined by high performance liquid chromatography. In six additional experiments, the vasodilator effect of 1 microM intracoronary adenosine was measured in the absence or presence of 100 or 200 nM XAC. RESULTS: Mild hypoxia decreased coronary resistance by 37 (SEM 4)% in the absence of XAC and 26(5)% or 17(4)% in the presence of 200 or 400 nM XAC, respectively. ISF[XAC] rapidly equilibrated with [XAC] in the arterial perfusate or venous effluent. XAC 400 nM markedly increased (p < 0.05) the hypoxic levels of venous and epicardial fluid adenosine from 49(19) and 251(42) nM to 75(11) and 495(48) nM, respectively. XAC 100-200 nM almost completely prevented the vasodilatation induced by 1 microM intracoronary adenosine. CONCLUSIONS: Adenosine mediates at least 54% of hypoxic vasodilatation. XAC rapidly equilibrates within the myocardial interstitial space and, as a result of blocking adenosine receptors, increases interstitial and venous adenosine concentrations. Increases in interstitial adenosine may partially overcome the adenosine receptor blockade by XAC, thereby reducing the effectiveness of XAC in attenuating the hypoxic vasodilatation. XAC attenuates intracoronary adenosine induced vasodilatation (mediated by endothelial adenosine receptors) much more effectively than it attenuates hypoxic vasodilatation, underscoring the minimal role played by the endothelial receptors in hypoxic vasodilatation.

Adenosine↗

PubMed1994

Nucleoside-induced arteriolar constriction: a mast cell-dependent response.

Adenosine (Ado) is a potent vasodilator that has occasionally been shown to cause vasoconstriction. Constrictor responses are generally attributed to A1-receptor stimulation or interactions with the renin-angiotensin system. We describe a previously unreported vasoconstrictor action of Ado and inosine (Ino) in hamster cheek pouch arterioles and examine the mechanism by which these nucleosides induce constriction. Arterioles were dissected from male Golden hamster cheek pouches, transferred to a 37 degrees C tissue chamber, and cannulated at both ends. Changes of luminal diameter in response to Ado were measured to generate cumulative concentration-response curves. The concentration-response curves were biphasic: 10(-6) M Ado elicited an intense, transient constriction, and higher concentrations induced dilator responses. Pretreatment with 8(p-sulfophenyl)theophylline, an Ado receptor antagonist, inhibited the dilator responses but did not alter the constriction. Inhibition of Ado uptake with S-(4-nitrobenzyl)-6-thio-inosine eliminated the constrictor response without altering dilator responses. Similar effects were found after pretreatment with an Ado deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Finally, Ino, a metabolite of Ado, induced constrictions of similar magnitude to those seen with Ado, but at higher concentrations. The constrictor response was focal in nature, suggesting discrete sites of action of Ado. Methylene blue staining after Ado application revealed degranulated mast cells closely associated with the vessel wall, indicating a possible role for mast cell degranulation in the constrictor response. Supporting this idea were the observations that inhibition of degranulation by 10 microM cromolyn blocked the constrictor response, and compound 48/80 (a mast cell secretagogue) caused constriction similar to that elicited by Ado.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed1994

Allosteric enhancer PD 81,723 acts by novel mechanism to potentiate cardiac actions of adenosine.

The 2-amino-3-benzoylthiophene derivative PD 81,723 is an allosteric enhancer of agonist binding to brain A1 adenosine receptors. One aim of this study was to characterize and contrast the effects of PD 81,723 on the A1 receptor-mediated negative dromotropic and A2a receptor-mediated vasodilatory actions of adenosine and of a nonmetabolizable and unselective N6-(3-pentyl)adenosine derivative. A second aim was to determine the mechanism of action of PD 81,723. In guinea pig isolated hearts, PD 81,723 potentiated the adenosine and the N6-(3-pentyl)adenosine derivative-induced prolongations of the stimulus-to-His bundle (S-H) interval in a concentration-dependent manner. PD 81,723 (30 mumol/L) decreased the EC50 value for adenosine to prolong the S-H interval by ninefold from 7.4 +/- 1.2 to 0.8 +/- 0.1 mumol/L but did not increase the content of adenosine in cardiac effluent. PD 81,723 (30 mumol/L) increased the specific binding of the A1 agonist [3H]cyclohexyladenosine ([3H]CHA) to human atrial and guinea pig atrial and brain membranes by 38%, 78%, and 300%, respectively. PD 81,723 also increased the fraction of A1 receptors in the high-affinity binding state by an average of 56 +/- 13%. The dissociation rate of [3H]CHA from guinea pig brain membranes was decreased in the presence of PD 81,723 (10 mumol/L) from 0.55 +/- 0.01/min to 0.35 +/- 0.01/min. PD 81,723 did not alter the binding of the A1 antagonist [3H]cyclopentyldipropylxanthine to guinea pig brain membranes. The IC50 values for 5'-guanylylimidodiphosphate to reduce specific binding of [3H]CHA to guinea pig cardiac and brain membranes were increased from 1.5 +/- 0.2 and 2.0 +/- 0.2 mumol/L in the absence of PD 81,723 to 10 +/- 3.3 and 18 +/- 0.5 mumol/L, respectively, in the presence of PD 81,723 (30 mumol/L). PD 81,723 did not potentiate the coronary vasodilatory actions of the N6-(3-pentyl)adenosine derivative. Specific binding of the A2a agonist [3H]CGS 21680 to brain membranes and the nucleoside transporter ligand [3H]nitrobenzylthioinosine to cardiac membranes was unchanged in the presence of PD 81,723. The results suggest that PD 81,723 specifically potentiates the action of adenosine on A1 receptors by stabilizing receptor-G protein interactions in the presence of agonists.

Adenosine↗

PubMed1993

Purification and partial amino acid sequence of a mu opioid receptor from rat brain.

A rat brain opioid receptor protein was isolated by binding [epsilon-biotinyl-Lys32] beta-endorphin to membranes, solubilizing the receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine and purifying on immobilized streptavidin and wheat germ agglutinin. The purified glycoprotein had a molecular mass of 60-70 kDa. Recovery of this protein was blocked by the nonselective opioid antagonist naloxone and the highly mu-selective agonist [D-Ala2,N-methyl-Phe4,Glyol5]-enkephalin but not by the highly delta-selective agonist [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin when these compounds were added as competitors at the binding step. The 60-70-kDa receptor protein co-purified through the streptavidin column with 40-kDa protein recognized by anti-Gi alpha antibodies. GTP and Na+ influenced dissociation of the solubilized R.125I-L complex and elution of the receptor and G protein from streptavidin in fashions consistent with the pharmacology of mu-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs at 4 positions from a similar sequence in the murine delta-opioid receptor and is encoded within a novel rat brain cDNA isolated by polymerase chain reaction with oligonucleotide primers related to the murine delta-opioid receptor gene.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and characterization of the human A3 adenosine receptor.

The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.

Adenosine↗

PubMed1993

Species comparison of adenosine and beta-adrenoceptors in mammalian atrial and ventricular myocardium.

The antagonist radioligand 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX) was used to characterize adenosine A1 receptors in membrane preparations from atrial and ventricular myocardium of rat, rabbit, guinea pig and pig. Kd values in crude membranes from guinea pig atria and ventricles (3.3 and 3.0 nM) were higher than those in the other species (ranges, 1.5-1.8 and 1.5-1.9 nM). Bmax values were greater in atria than in ventricles in all four species, and in atria and ventricles of guinea pig (76 and 34 fmol/mg), than in the other species (ranges, 15-17 and undetectable to 12 fmol/mg). In contrast, guinea pig Kd and Bmax values for beta-adrenoceptors, which were labelled with (-)3-[125I]iodocyanopindolol, fell within the range of values for the other three mammalian species. In semipurified membrane preparations from pig, [3H]DPCPX and the agonist radioligand [125I]-N6-4-aminobenzyladenosine appeared to label a similar population of receptors and gave comparable Kd values in atria (0.73 and 0.66 nM) and in ventricles (0.57 and 0.70 nM). In semipurified preparations from pig, the agonist R-(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) displaced [3H]DPCPX in a manner consistent with the presence of both high- and low-affinity adenosine A1 receptors. The data from this study indicate that the density of adenosine A1 receptors in atria is greater than in ventricles, but similar Kd values suggest that the A1 receptor population is the same in the two cardiac tissues. Also, the data demonstrate that the [3H]DPCPX antagonist binding characteristics of guinea pig myocardium differ from those in rat, rabbit and pig.

Adenylyl Cyclases↗

PubMed1993

Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor.

Somatostatin (SRIF) receptors of GH4C1 cells occupied with biotinyl-NH-[Leu8,D-Trp22,Tyr25] somatostatin28 (bio-S28) have been affinity purified over streptavidin affinity columns (Eppler, C. M., Zysk, J. R., Corbett, M., and Shieh, H.-M. (1992) J. Biol. Chem. 267, 15603-15612). This procedure results in the copurification of a single subtype of SRIF receptor (SSTR2) and associated guanine nucleotide-binding proteins (G proteins) that are coupled to these receptors. For accurate quantification it was necessary to: (i) use homogenous recombinant standards; (ii) accurately assess the purity of standards; (iii) determine recovery of G proteins during sample preparation and Western blotting; and (iv) account for cross-reactivity among antisera. Four pertussis toxin-sensitive G proteins were quantified with previously characterized polyclonal antisera. Gi alpha 1 also was measured with a novel, more sensitive monoclonal antibody (7H7). Go alpha and Gi alpha 2 but not Gi alpha 1 and Gi alpha 3 were detected in membrane extracts prepared from GH4C1 cells. In contrast, the G proteins copurified with SSTR2 receptors were predominantly Gi alpha 2 (50% of total G protein) and Gi alpha 3 (36% of total G protein), whereas Go alpha and Gi alpha 1 were negligible. G beta subunits also were detected. Silver staining confirmed the absence of a 39-kDa protein, corresponding to the M(r) of Go alpha associated with purified SRIF receptor-G protein complexes. These data suggest that SRIF receptors selectively couple to two G proteins, one of which is sparsely expressed in GH4C1 cells; the data conform to the notion that SRIF receptors discriminate between similar pertussis toxin-sensitive G proteins.

Amino Acid Sequence↗

PubMed1993

Cloned receptors and cardiovascular responses to adenosine.

Four subtypes of adenosine receptors have recently been cloned from thyroid, brain and testis. In this review we have summarised properties of these purinergic receptors. The cloned A1 and A2 subtypes are probably similar or identical to receptors that exist on cardiac and vascular tissues, respectively. A comparison of the amino acid sequences of A1, A2a, and A2b receptors reveals several stretches of conserved amino acids that are unique to adenosine receptors, primarily in the membrane spanning regions. Species differences in A1 receptors indicate that minor changes in receptor structure can produce marked changes in ligand binding properties and may facilitate the identification of amino acids involved in ligand recognition. A confusing A1 receptor subclassification system of putative A1a, A1b, and A3 subtypes has emerged based on subtle rank order potency differences for various ligands among tissues. cDNAs corresponding to these A1 subtypes have not yet been isolated. Atrial A1 receptors activate K+ channels and inhibit adenylyl cyclase. These two pathways appear to be independently up and down regulated, suggesting the existence either of atrial A1 receptor subtypes or of differential regulation of the coupling of a single receptor to distinct GTP binding proteins. An adenosine receptor distinct from A1 and A2 receptors has been cloned from testis and designated TGPCR, or A3, although it differs from the pharmacologically defined A3 receptor. We suggest that the current A1/A3 receptor subtype nomenclature be abandoned and superseded by a nomenclature based solely on receptor cDNAs. In addition to the cloned adenosine receptors, a novel A4 subtype has been proposed based on pharmacological and electrophysiological criteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1993

Adenosine stimulates human sperm motility via A2 receptors.

The effects of adenosine and its analogues on human sperm motility were studied using a transmembrane migration method. Specific binding sites for adenosine in human sperm were also investigated. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) stimulated human sperm motility with similar efficacies and the maximal amplitudes of motility increases were both about 70%. 3,7-Dimethyl-1-propargylxanthine (DMPX), a potent A2 antagonist, competitively antagonized NECA-induced motility stimulation. Successively higher concentrations of DMPX shifted the dose-response curve of NECA to the right in a nearly parallel fashion. Dipyridamole, an inhibitor of adenosine uptake, does not reduce the ability of adenosine to stimulate human sperm motility. In radioligand-binding studies, adenosine A1 selective analogues, cyclopentyl-1,3-dipropylxanthine and 1-methyl-2-phenylethyl adenosine, have little competitive effect on [3H]NECA binding in human sperm membrane. These results provide evidence that adenosine enhances human sperm motility via adenosine A2 receptors on the surface of sperm membranes.

Adenosine↗

PubMed1993

Differential sensitization of cardiac actions of adenosine in rats after chronic theophylline treatment.

To determine the effect of chronic adenosine receptor blockade on atrial responsiveness, we administered theophylline to rats in their drinking water (0.6 mg/ml) for 2 wk. Inotropic and chronotropic responses to the adenosine receptor agonists N6-cyclopentyladenosine (CPA) and 5'-(N-ethylcarboxamido)-adenosine (NECA) were then measured in isolated atria from treated and control animals. The indirect (antiadrenergic) actions of CPA and NECA on force and rate, measured during beta-adrenergic receptor stimulation by isoproterenol, were markedly sensitized (2- to 10-fold reductions in the agonist concentration needed to obtain a half-maximal response) after theophylline. The sensitization was homologous because inotropic and chronotropic responses to carbachol were not affected by theophylline. The direct negative inotropic and chronotropic actions of CPA and NECA, measured without concomitant beta-adrenergic stimulation, were not sensitized after theophylline. The number of atrial A1-receptors, measured by antagonist radioligand binding (maximum specific binding at saturation), was increased by 22% in theophylline-treated rats [66.2 +/- 3.4 vs. 54.3 +/- 1.9 (control) fmol/mg protein, P < 0.05]. Competition binding indicated that the fraction of coupled (high-affinity) receptors was unchanged. The number of ventricular A1-receptors was increased to a similar extent without any change in coupling. Thus chronic dietary theophylline upregulated cardiac A1-adenosine receptors without changing coupling state or affinity and sensitized rat atria to the indirect, antiadrenergic, inhibitory inotropic and chronotropic actions of adenosine receptor agonists.

Adenosine↗

PubMed1993

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

Amino Acid Sequence↗

PubMed1992

Solid phase enzyme immunoassay of cyclic adenosine 3',5'-monophosphate. Effect of coating strategy upon assay performance in comparison with radioimmunoassay.

We have evaluated two novel enzyme-linked immunosorbent assays (ELISAs) used to quantitate cyclic AMP. In one assay ELISA plates are coated with antigen consisting of a cyclic AMP-polylysine conjugate. Cyclic AMP samples added to plates are quantified by their ability to decrease the binding and anti-cyclic AMP antibodies to the coated antigen. A second ELISA utilizes a plated anti-immunoglobin technique in which plates are coated first with anti-goat IgG and then with goat anti-cyclic AMP antiserum. Cyclic AMP samples are quantified by their ability to compete with cyclic AMP-peroxidase conjugates for binding to the plated anti-cyclic AMP antibodies. The plated anti-immunoglobin ELISA proved to be somewhat more sensitive than the plated antigen ELISA and was comparable in sensitivity to an automated RIA for measuring cyclic AMP in standards and urine samples. Our data fit with the generalization that optimal ELISA sensitivity is obtained through the use of plated anti-immunoglobins rather than plated antigens. Further they demonstrate the practicality of utilizing small ligand-enzyme conjugates for ELISAs.

Cyclic AMP↗

PubMed1992

Cloning and expression of a bovine adenosine A1 receptor cDNA.

A bovine brain adenosine A1 receptor cDNA encoding a 326 amino acid protein has been identified. This cDNA, which encodes a protein greater than 90% identical to analogous rat and dog receptors, was transiently expressed in COS-1 cells. Recombinant receptors exhibited the features of bovine A1 receptors that distinguish it from rat and canine receptors, including subnanomolar Ki for 1,3-dipropyl-8-cyclopentylxanthine, R-phenylisopropyl- adenosine (R-PIA) and xanthine amino conjugate, and the distinct potency order: R-PIA greater than S-PIA much greater than 5'-N-ethylcarboxamidoadenosine greater than 2'-chloroadenosine. The results indicate that the pharmacological differences between A1 adenosine receptors among species result from only minor differences in receptor structures.

Amino Acid Sequence↗

PubMed1992

The precise radioimmunoassay of adenosine: minimization of sample collection artifacts and immunocrossreactivity.

Anti-adenosine antibodies were produced in rabbits immunized with N6-carboxymethyladenosine conjugated to methyl albumin. 125I-N6-Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 nM (312.5 fmol) of underivatized adenosine and cross-reacts less than 0.02% with adenine nucleotides and guanosine and not at all with 1 mM inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 nM (6.25 fmol) by derivitizing samples with benzyl bromide to form N6-benzyladenosine. The assay was adapted to an automated RIA procedure. Assay precision was increased by: (i) inhibiting slight adenosine deaminase activity present in anti-sera; (ii) treating buffers and albumin used in the RIA with charcoal to remove contaminating adenosine; and (iii) correcting for a small but variable component of immunoreactivity not attributable to adenosine. A second antibody prepared with a 2',3'-disuccinyladenosine-albumin conjugate was also found to detect some non-adenosine-mediated immunoreactivity in plasma samples. Immunointerference in human plasma was eliminated in samples treated with ZnSO4/Ba(OH)2 or partially purified over C18 Sep Paks to remove nucleotides and assayed after sample benzylation or succinylation. Human blood was mixed with a novel "stop" solution that was optimized to inhibit adenosine formation from AMP by greater than 99% and to inhibit adenosine uptake into red cells and degradation by greater than 94%. Human plasma/stop solution was assayed by RIA and HPLC with equivalent results.

Adenine↗

PubMed1992

Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C.

The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family.

Adenosine Triphosphate↗

PubMed1992

Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine.

Guanine nucleotides such as guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) have been found to increase the binding of antagonists to adenosine A1 receptors. This response can be attributed either to a direct effect of GTP on receptors to increase antagonist affinity or to an indirect effect to decrease the affinity of receptors for a pool of endogenous adenosine that cannot be readily removed from membranes. In this study, adenosine content was measured in preparations of membranes and 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS)-solubilized receptors by a sensitive radioimmunoassay. In both preparations, pools of adenosine (2.5-10 pmol/mg of protein) were detected that were resistant to deamination by added adenosine deaminase (0.5-3 units/ml) unless membrane lipids were first dissolved in acetone. Electron microscopic examination of crude CHAPS-solubilized receptors revealed the existence of small vesicles (< 1 microns in diameter). Furthermore, most "solubilized" receptors were retained by a 0.1-microns filter. The effects of GTP gamma S were evaluated on the binding of an antagonist, 3-(4-amino-3-125I-phenethyl)-1-propyl-8-cyclopentylxanthine (125I-BW-A844U), to A1 receptors of bovine brain membranes, receptors solubilized in CHAPS (crude solubilized), or receptors partially co-purified with G proteins by agonist affinity chromatography (partially purified). GTP gamma S (10 microM) increased antagonist binding to membranes (20-50%) and crude CHAPS-solubilized receptors (> 200%) but increased binding to partially purified receptors by only 10-15%. GTP gamma S decreased agonist (125I-N6-aminobenzyladenosine) binding and increased antagonist Bmax, but did not significantly decrease (5%) the dissociation rate of the antagonist. Omission of Mg2+ mimicked the effects of GTP gamma S on agonist and antagonist binding and increased both the association and dissociation rates of 125I-BW-A844U. These data suggest that a Mg(2+)-dependent GTP gamma S-induced increase in antagonist binding to membranes and solubilized receptors is primarily due to unmasking of cryptic binding sites occupied by contaminating vesicular adenosine. These findings are consistent with the observation that adenosine receptor antagonists have been found to have little or no inverse agonist physiological effects in well oxygenated tissues.

Adenosine↗

PubMed1992

Improved detection of ischemia-induced increases in coronary sinus adenosine in patients with coronary artery disease.

Attempts to monitor coronary sinus adenosine as a clinical marker of myocardial ischemia in humans have been disappointing. Accordingly, procedures have been developed for detecting adenosine in blood collected from the human coronary sinus. Collection involves using a double-lumen metabolic catheter, which allows blood to be mixed with a stop solution at the catheter tip, thereby minimizing adenosine formation and degradation. A five-component stop solution almost completely arrests adenosine formation and degradation. Adenosine analysis is improved by using both boronate and C18 Sep-Pak columns to purify and concentrate adenosine in human plasma before HPLC. Plasma adenosine in the coronary sinus of patients with and without coronary artery disease, measured before and during peak atrial pacing, showed a twofold atrial pacing-induced increase in adenosine in the patients with coronary artery disease (n = 9, P less than 0.001) but no change in the patients with normal epicardial coronary arteries (n = 6). These preliminary results indicate that coronary sinus adenosine may provide an index of myocardial ischemia in patients with coronary artery disease.

Adenosine↗

PubMed1991

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go.

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.

Animals↗