Therapeutic drug monitoring of levetiracetam: Method validation using high-performance liquid chromatography-ultraviolet detector technique and usefulness in patient care setting.

Objectives: To develop and validate a modified HPLC-UV method for the estimation of serum levetiracetam levels and to assess the usefulness of serum levetiracetam estimation in epileptic patients. Materials and Methods: Modification of a previously existing HPLC-UV method was performed using liquid- liquid phase extraction and processing using reverse phase analytic HPLC-UV detector technique followed by method validation. Serum samples of patients attending our hospital's Therapeutic Drug Monitoring Outpatient Department services were analyzed for levetiracetam levels using the study method. Data of the past 6 years (2015-2020) were descriptively analyzed. Results: The modified HPLC-UV method was validated as per ICH Q2 (R1) 2005 guidelines. Usefulness of levetiracetam estimation was assessed in 1383 patients (635 children, 683 adults, 40 elderly, and 25 pregnant women). Levetiracetam levels were within the therapeutic range (TR) in 520 children, 543 young adults, 35 elderly patients, and nine pregnant women. In 112 of 232 patients with low levetiracetam levels, poor compliance was elicited. Of 641 patients on polytherapy, 446 patients had levetiracetam values within TR, whereas 29 had values above and 166 patients had values less than TR. Sodium valproate, phenytoin sodium, and carbamazepine affected levetiracetam levels when given concomitantly. Levetiracetam dose was adjusted in 61 patients with abnormal levels for better clinical response. Good seizure control was noted in 913 (82.47%) patients whose levels were within TR, whereas 136 (58.62%) patients with low levels reported an increase in seizure frequency. Conclusions: The modified HPLC-UV method is simple, rapid, efficient, and reliable for assaying serum levetiracetam.

Plumbagin exerts antiobesity effects through inhibition of pancreatic lipase and adipocyte differentiation.

Plumbagin is a naphthoquinone found in the roots of Plumbago zeylanica. Here, we report an investigation to evaluate its antiobesity activity. The preliminary binding affinity of plumbagin to human pancreatic lipase (PL) was determined using molecular docking simulation. The in vitro PL inhibitory potential and the kinetics of inhibition were studied to validate and confirm the results obtained from molecular docking. The IC50 for PL was found to be 82.08 ± 9.47 µM, and the kinetics of inhibition was found to be of the mixed type. Further, the in vivo evaluation revealed that rats treated with plumbagin 1 mg/kg showed significant decrease in serum triglycerides (TG) and area under the curve of serum TG when compared with vehicle-treated rats. It was also seen that plumbagin possessed significant antiadipogenic effect as demonstrated by reduced oil red O staining and decreased TG contents. Thus, we conclude that plumbagin is a promising molecule to combat obesity and further optimization of plumbagin to yield plumbagin analogues will result in its improved activity profile.

Review of policies for injuries to research participants in India.

BACKGROUND: As there is little Indian data about severity, frequency and types of research related injuries, costs involved and policies regarding compensation, this study was conducted to review the present Indian scenario. METHODS: The study was carried out in three parts; a questionnaire-based survey, in-depth interviews, and a review of informed consent and insurance documents of projects submitted to three ethics committees. RESULTS: 47% of investigators were either unaware of, or had not understood, the legal requirements and depended on sponsors to manage these issues, whereas 74% of ethics committee members were aware of the requirements. Although 40% of investigators, 30% of ethics committee members and all sponsors had policies to manage compensation issues, these were mainly to provide immediate free medical care or reimbursement of expenses incurred for the acute management of an adverse event. Compensation for loss of time/wages, death, physical disability or long term incapacitation was not included. A review of informed consent and insurance documents showed that compensation issues were inadequately discussed, with only insurance certificates submitted to ethics committees. CONCLUSION: In India, there are no uniform policies and investigators are largely unaware of their responsibilities. Therefore, there is an urgent need to draft national guidelines regarding compensation for research injuries of research participants and highlight the responsibilities of each stakeholder. Potential research injuries should be categorised based on risk assessment, severity and seriousness of the injury. Further, it would be necessary to have arbitration committees to determine the extent of compensation. Training and awareness workshops for those involved in clinical research, including research participants, is also needed.

Identification of a role for actin in translational fidelity in yeast.

Numerous studies have suggested a role for actin in translation, but the molecular details of this role are unknown. To elucidate the function(s) of actin in translation, we have studied 25 isogenic, conditional yeast actin mutants. Strikingly, analysis of these mutants indicates that none of those tested have conditional growth defects caused by reduced rates of protein synthesis; and analysis of latrunculin A-treated wild-type cells indicates that even complete disruption of the actin cytoskeleton has no significant effect on the rate of translation. However, analysis of the effect of the 25 actin mutations on fidelity and sensitivity to translation inhibitors identified two mutations ( act1-2 and act1-122) that cause a significant reduction in the fidelity of translation, as assayed by nonsense suppression, and several mutants that are sensitive to paromomycin, which affects translational fidelity. Translation elongation factor 1A (eEF1A) also has a role in fidelity, and in the presence of excess eEF1A four of the mutants ( act1-2, act1-20, act1-120, and act1-125) are even more sensitive to paromomycin, while one mutant ( act1-122) becomes less sensitive. Together, these findings suggest that actin may not be important for the rate of translation, but may have a critical role in ensuring translational fidelity.

Characterization of a general stabilizer element that blocks deadenylation-dependent mRNA decay.

mRNA degradation is a regulated process that can play an important role in determining the level of expression of specific genes. The rate at which a specific mRNA is degraded depends largely on specific cis-acting sequences located throughout the transcript. cis-Acting destabilizer sequences that promote increased rates of decay have been identified in several short-lived mRNAs. However, little is known about elements that promote stability, known as stabilizer elements (STEs), and how they function. The work presented here describes the characterization of a STE in the PGK1 transcript. The PGK1 stabilizer element (P-STE) has been delineated to a 64-nucleotide sequence from the coding region that can stabilize a chimeric transcript containing the instability elements from the 3'-untranslated region of the MFA2 transcript. The P-STE is located within the PGK1 coding region and functions when located in the translated portion of the transcript and at a minimum distance from the 3'-untranslated region. These results further support the link between translation and mRNA degradation. A conserved sequence in the TEF1/2 transcript has been identified that also functions as a STE, suggesting that this sequence element maybe a general stability determinant found in other yeast mRNAs.

Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast.

The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

Replication of nuclei from cycling and quiescent mammalian cells in 6-DMAP-treated Xenopus egg extract.

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei in Xenopus egg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [alpha-32P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the "licensed state" of G0 nuclei using the assays described here.

Reactivation of DNA replication in nuclei from terminally differentiated cells: nuclear membrane permeabilization is required for initiation in Xenopus egg extract.

We have used Xenopus egg extract to investigate the requirements for reactivation of DNA replication in nuclei isolated from terminally differentiated chicken erythrocytes. Previous work has shown that reactivation of erythrocyte nuclei in egg extract is accompanied by chromatin decondensation, nuclear envelope reformation, and the accumulation of egg lamin, LIII. However, in those studies, erythrocyte nuclei were prepared by methods that were not designed to maintain the selective permeability of the nuclear membrane, and as such, it is not clear if loss of nuclear membrane integrity played a role in the reactivation process. Therefore, the purpose of this study was to determine if changes in nuclear membrane permeability are required for reactivation of erythrocyte nuclei in egg extract. Nuclei with intact nuclear membranes were prepared from erythrocytes with streptolysin O and permeable nuclei by treatment of intact nuclei with the detergent Nonidet-P40. Like permeable nuclei, most intact nuclei decondensed, imported nuclear protein, and accumulated lamin LIII from the extract. However, unlike permeable nuclei, which replicated extensively in the extract, few intact nuclei initiated replication under the same conditions. These data demonstrate that permeabilization of the nuclear membrane is required for reactivation of DNA replication in terminally differentiated erythrocyte nuclei by egg extract and suggest that loss of nuclear membrane integrity may be a general requirement for replication of quiescent cell nuclei by this system.

Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract.

We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.

Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane.

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact-inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact-inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha-32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.

P2U-purinergic receptors on C6-2B rat glioma cells: modulation of cytosolic Ca2+ and cAMP levels by protein kinase C.

The activation of P2-purinergic receptors on C6-2B rat glioma cells caused a transient increase in cytosolic-free Ca2+ concentration ([Ca2+]i) as detected by Fura 2 fluorescence ratio imaging of single cells. These purinergic receptors are of the P2U subtype because UTP and ATP were equipotent and substantially more potent than the P2X- and P2Y-selective agonists alpha,beta-methylene ATP and 2-methylthio ATP, respectively. There was homologous desensitization of the Ca2+ responses between UTP and ATP but no heterologous desensitization between these nucleotides and another Ca(2+)-mobilizing receptor agonist, alpha-thrombin. The UTP-induced peak [Ca2+]i rise was insensitive to chelation of extracellular Ca2+ with EGTA. However, the response was abolished after either depletion of intracellular Ca2+ stores with the microsomal Ca(2+)-ATPase inhibitor thapsigargin or blockade of Ca2+ release from intracellular stores with the muscle relaxant dantrolene. The activation of P2U-purinergic receptors and thrombin receptors increased the formation of total inositol phosphates (IPs) and inhibited cAMP accumulation elicited with either the beta-adrenergic receptor agonist (-)-isoproterenol, or forskolin, a direct activator of adenylyl cyclase. UTP- and alpha-thrombin-induced changes in the levels of IPs, cytosolic Ca2+, and agonist-elicited cAMP accumulation were dramatically inhibited (> 80%) by acute treatment of the cells with the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate but not with the inactive ester 4 alpha-phorbol 12,13-didecanoate. We conclude that in C6-2B cells, the increase in [Ca2+]i after activation of P2U-purinergic receptors is primarily a result of IPs-mediated release of Ca2+ from intracellular stores with secondary influx of Ca2+ by capacitative mechanisms. Also, the inhibition by UTP and alpha-thrombin of agonist-elicited cAMP accumulation is mediated through an increase in [Ca2+]i.

Predominant expression of type-VI adenylate cyclase in C6-2B rat glioma cells may account for inhibition of cyclic AMP accumulation by calcium.

In C6-2B cells, agonist-stimulated cyclic AMP accumulation is inhibited when the cytosolic Ca2+ concentration is increased. We now demonstrate that in C6-2B cells: (i) the early kinetics of the cyclic AMP inhibition by substance K (t1/2 = 35 s) and thapsigargin (t1/2 = 1.6 min) closely mimic the kinetics of the cytosolic Ca2+ increase evoked by either agent (t1/2 = 25 s and 1.5 min respectively); (ii) the Ca2+ rise and cyclic AMP inhibition by substance K or thapsigargin are similarly affected in EGTA-containing medium; (iii) PCR detects type-III and type-VI adenylate cyclase cDNAs, and RNAase protection assays show that the mRNA for type-VI adenylate cyclase, an isoform inhibitable by submicromolar Ca2+ concentrations, is the predominant species, strongly suggesting that type-VI adenylate cyclase is probably the target molecule for Ca(2+)-mediated inhibition of cyclic AMP accumulation.

Ca2+ inhibition of beta-adrenergic receptor- and forskolin-stimulated cAMP accumulation in C6-2B rat glioma cells is independent of protein kinase C.

In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.

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.

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.

Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution.

A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.

Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain.

A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.

5'-Deoxy-5'-methylthioadenosine: a nucleoside which differentiates between adenosine receptor types.

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.

[125I]N6-(3-iodo-4-hydroxyphenyl)isopropyladenosine: the use of the diastereomers as ligands for adenosine receptors in rat brain.

The diastereomers of N6-(4-hydroxyphenyl)isopropyladenosine (HPIA) were synthesized, radioiodinated and used as ligands for adenosine receptors in rat brain membranes. In contrast to the S-isomer, specific binding (measured by displacement with 10 microM N6-R-phenylisopropyladenosine (PIA] of [125I]N6-[R-(3-iodo-4-hydroxyphenyl)isopropyl] adenosine ([125I]R- IHPIA) required about 2 h for equilibrium but bound with a higher affinity. Scatchard analysis of binding data were compatible with the existence of single binding site with Kd values of 0.7 nM (R-isomer) and 10 nM (S-isomer), and maximal binding of 228 and 237 fmol/mg, respectively, at 30 degrees C. Computer-based non-linear curve fitting resulted in estimates of 0.67 and 7.5 nM, and of 208 and 173 fmol/mg, for the R- and S-isomer, respectively. Dissociation of the R-isomer in the presence of 10 microM PIA was biphasic, both phases being increased in the presence of 100 microM Gpp(NH)p. The ratio of the rate of dissociation for the slower phase, k2, and the rate of association, k1, yielded a further estimate for Kd of 0.3 nM. The specific binding of [125I]R-IHPIA was displaced by adenosine receptor agonists with the following order of decreasing potency: N6-[R-(phenyl)-isopropyl]adenosine = N6-[R-(4-hydroxyphenyl)isopropyl]adenosine greater than 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine greater than N6-[S-(phenyl)isopropyl]adenosine = N6-[S-(4-hydroxyphenyl)isopropyl]adenosine which is typical of binding to Ri/A1 type receptors. Receptor antagonists displaced this binding in the following order of decreasing potency: 8-(p-hydroxyphenyl) theophylline greater than 8-phenyltheophylline greater than theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)