Biomedical subjects

M F Jarvis

Publications and source records attributed to M F Jarvis.

At least 37 records · Page 2Linked to original sources

PubMed2000

ABT-702 (4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin- 3-yl)pyrido[2,3-d]pyrimidine), a novel orally effective adenosine kinase inhibitor with analgesic and anti-inflammatory properties. II. In vivo characterization in the rat.

Adenosine kinase (AK; EC 2.7.1.20) is a key intracellular enzyme regulating intra-and extracellular concentrations of adenosine (ADO), an endogenous neuromodulator, antinociceptive, and anti-inflammatory autocoid. AK inhibition provides a means of potentiating local tissue concentrations of endogenous ADO, and AK inhibitors may have therapeutic potential as analgesic and anti-inflammatory agents. The effects of ABT-702, a novel, potent (IC(50) = 1.7 nM), and selective non-nucleoside AK inhibitor were examined in rat models of nociception and acute inflammation. ABT-702 was orally effective and fully efficacious to suppress nociception in a spectrum of pain models in the rat, including carrageenan-induced thermal hyperalgesia, the formalin test of persistent pain, and models of nerve injury-induced and diabetic neuropathic pain (tactile allodynia after L5/L6 spinal nerve ligation or streptozotocin injection, respectively.) ABT-702 was especially potent at relieving inflammatory thermal hyperalgesia (ED(50) = 5 micromol/kg p.o.). ABT-702 was also effective in the carrageenan-induced paw edema model of acute inflammation (ED(50) = 70 micromol/kg p.o.). The antinociceptive and anti-inflammatory effects of ABT-702 were blocked by selective ADO receptor antagonists, consistent with endogenous ADO accumulation and ADO receptor activation as a mechanism of action. The antinociceptive effects of ABT-702 were not blocked by the opioid antagonist naloxone. In addition, ABT-702 showed less potential to develop tolerance to its antinociceptive effects compared with morphine. ABT-702 had no significant effect on rotorod performance or heart rate (at 30-300 micromol/kg p.o.), mean arterial pressure (at 30-100 micromol/kg p.o.), or exploratory locomotor activity (at </=10 micromol/kg p.o.). Thus, ABT-702 is a novel, non-nucleoside AK inhibitor, with a nonopioid, non-nonsteroidal anti-inflammatory drug mechanism of action, which shows antinociceptive and anti-inflammatory activity in vivo.

Adenosine Kinase↗

PubMed1999

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A(1) receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [3H]cyclohexyladenosine ([3H]CHA) to A(1) receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81, 723 (0.3-100 microM) produced a concentration-dependent enhancement of [3H]CHA binding to rat brain A(1) receptors. These effects were also species-dependent with larger enhancements (150-200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [3H]CHA binding to A(1) receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 microM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A(1) receptor selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 microM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [3H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 microM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A(1) receptor in a high-affinity state and that the relative proportions of high-affinity A(1) receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A(1) receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A(1) receptor.

Adenosine↗

PubMed1999

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Adenosine Triphosphate↗

PubMed1999

Site and event specific increase of striatal adenosine release by adenosine kinase inhibition in rats.

The effects of the systemically administered adenosine kinase (AK) inhibitor, 5'-deoxy-5-iodotubercidin (5'd-5IT) on the striatal adenosine (ADO) release evoked by the excitotoxin, kainic acid (KA) were examined using rat bilateral striatal microdialysis. Local KA perfusion of one rat striatum caused a significant ipsilateral elevation of striatal ADO levels compared to basal and contralateral (artificial CSF-perfused) striatal ADO levels. KA-evoked striatal ADO release was augmented in animals receiving systemic 5'd-5IT treatment (cumulative dose of 7.5 micromol/kg, i.p.) compared with i.p. vehicle controls. In contrast, 5'd-5IT administration had no significant effect on basal or contralateral (artificial CSF-perfused) striatal ADO levels. Thus, consistent with the hypothesis of 'site and event specific' potentiation of ADO by AK inhibitors, 5'd-5IT unilaterally enhanced ADO levels in the striatum where KA-induced excitotoxic injury evoked endogenous ADO release, but not at the contralateral uninjured striatum.

Adenosine↗

PubMed1999

An adenosine kinase inhibitor attenuates tactile allodynia in a rat model of diabetic neuropathic pain.

The present study was conducted to characterize the development of tactile allodynia in the streptozotocin-induced rat model of diabetes, and to evaluate the antinociceptive effects of systemically administered morphine and the adenosine kinase inhibitor, 5'-deoxy-5-iodotubercidin (5'd-5IT) in this model. Rats were injected with 75 mg/kg streptozotocin (i.p.), and blood glucose levels were determined 3-4 weeks later. Diabetic (blood glucose levels > or = 250 mg/dl) and vehicle-injected rats were examined weekly for the development of tactile allodynia by measuring the threshold for hind paw withdrawal using von Frey hairs. Withdrawal thresholds were reduced to 6.8+/-0.6 g (mean+/-S.E.M.) in approximately one-third of streptozotocin-treated rats 7 weeks after streptozotocin treatment as compared to control thresholds (13.2+/-0.1 g), and this allodynia persisted for at least an additional 7 weeks. In additional experiments, morphine sulfate (5-21 micromol/kg, i.p.) produced dose-dependent antinociceptive effects on tactile allodynia for up to 2 h post-dosing. The adenosine kinase inhibitor, 5'd-5IT (2.5 and 5 micromol/kg, i.p.) also dose-dependently attenuated tactile allodynia. Pretreatment with the opioid receptor antagonist, naloxone (27 micromol/kg, i.p.) or the non-selective adenosine receptor antagonist, theophylline (111 micromol/kg, i.p.) significantly diminished the anti-allodynic effects of morphine and 5'd-5IT, respectively. The present study demonstrates that the potent and selective adenosine kinase inhibitor, 5'd-5IT, is equally effective as morphine in blocking tactile allodynia in this model.

Adenosine Kinase↗

PubMed1999

Characterization of the effects of adenosine kinase inhibitors on acute thermal nociception in mice.

Adenosine (ADO) is an inhibitory neuromodulator that can increase the nociceptive threshold in animals exposed to a variety of noxious stimuli. Inhibition of the ADO-metabolizing enzyme, ADO kinase (AK), provides a means of locally enhancing extracellular ADO concentrations. In the present study, the AK inhibitors 5'-amino,5'-deoxy-ADO (NH2dADO), 5-iodotubercidin (5-IT), and 5'-deoxy,5-iodotubercidin (5'd-5IT) were examined for their analgesic efficacy in the hot-plate model of acute somatic nociception. Control and drug-treated adult male mice were placed on a 55 degrees C hot plate and the latency to the 10th jump was recorded via a computer driven infrared-beam photosensor. All three AK inhibitors were found to significantly increase jump latencies in a dose-dependent fashion. 5'd-5IT was the most potent AK inhibitor (approx. ED50 value = 1 micromol/kg, IP), followed by 5-IT (ED50 value = 10 micromol/kg, IP), and NH2dADO (ED50 value = 100 micromol/kg, IP). 5'd-5IT was found to be more potent and equally efficacious to morphine (ED50 value = 5.2 micromol/kg, IP) in this assay. In a model of persistent chemical pain, the phenyl-p-quinone-induced abdominal constriction assay, 5'd-5IT (ED50 value = 1.5 micromol/kg, SC) and morphine (ED50 value = 3.0 micromol/kg, SC) dose dependently reduced nociception. Pretreatment of mice with either the nonselective ADO receptor antagonist, theophylline (56 micromol/kg, IP), but not the peripherally acting antagonist, 8-(p-sulfophenyl)-theophylline (8-PST, 200 micromol/kg, IP) significantly attenuated the antinociceptive effects of 5'd-5IT in the hot-plate assay. Furthermore, the antinociceptive effects of 5'd-5IT were completely blocked by an ADO A1 receptor selective antagonist, DPCPX, while an ADO A2A receptor selective antagonist, ZM 241385, showed markedly less antagonist activity. The analgesic effects of 5'd-5IT were not blocked by the opioid receptor antagonist naloxone; however, 5'd-5IT could produce additive analgesic effects with morphine when both compounds were administered in combination. The apparent efficacy of 2.5 micromol/kg, IP, of 5'd-5IT was not significantly altered following the repeated administration of this dose twice daily for 4 days. The present data provide evidence for an antinociceptive action of AK inhibitors in the hot-plate test, which, at least for 5'd-5IT, is mediated by an enhancement of ADO's actions at the ADO A1 receptor subtype, is nonopioid in nature, and which does not exhibit tolerance following repeated administration.

Adenosine Kinase↗

PubMed1999

Molecular and functional characterization of human P2X(2) receptors.

P2X receptors are a family of ATP-gated ion channels. Four cDNAs with a high degree of homology to the rat P2X(2) receptor were isolated from human pituitary and pancreas RNA. Genomic sequence indicated that these cDNAs represent alternatively spliced messages. Northern analysis revealed high levels of human P2X(2) (hP2X(2)) message in the pancreas, and splice variants could be detected in a variety of tissues. Two cDNAs encoded functional ion channels when expressed in Xenopus oocytes, a receptor structurally homologous to the prototype rat P2X(2) receptor (called hP2X(2a)) and a variant containing a deletion within its cytoplasmic C terminus (called hP2X(2b)). Pharmacologically, these functional human P2X(2) receptors were virtually indistinguishable, with the P2X receptor agonists ATP, 2-methylthio-ATP, 2' and 3'-O-(4-benzoylbenzoyl)-ATP, and ATP5'-O-(3-thiotriphosphate) being approximately equipotent (EC(50) = 1 microM) in eliciting extracellular Ca(2+) influx. The P2 receptor agonists alpha,beta-methylene ATP, adenosine, adenosine 5'-O-(2-thiodiphosphate), and UTP were inactive at concentrations up to 100 microM. Both hP2X(2a) and hP2X(2b) receptors were sensitive to the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (IC(50) = 3 microM). In contrast to the analogous rat P2X(2) and P2X(2b) receptors, the desensitization rates of the hP2X(2a) and hP2X(2b) receptors were equivalent. Both functional forms of the human P2X(2) receptors formed heteromeric channels with the human P2X(3) receptor. These data demonstrate that the gene structure and mRNA heterogeneity of the P2X(2) receptor subtype are evolutionarily conserved between rat and human, but also suggest that alternative splicing serves a function other than regulating the desensitization rate of the human receptor.

Adenosine Triphosphate↗

PubMed1999

P2X receptor-mediated ionic currents in dorsal root ganglion neurons.

Nociceptive neurons in the dorsal root ganglia (DRG) are activated by extracellular ATP, implicating P2X receptors as potential mediators of painful stimuli. However, the P2X receptor subtype(s) underlying this activity remain in question. Using electrophysiological techniques, the effects of P2X receptor agonists and antagonists were examined on acutely dissociated adult rat lumbar DRG neurons. Putative P2X-expressing nociceptors were identified by labeling neurons with the lectin IB4. These neurons could be grouped into three categories based on response kinetics to extracellularly applied ATP. Some DRG responses (slow DRG) were relatively slowly activating, nondesensitizing, and activated by the ATP analogue alpha,beta-meATP. These responses resembled those recorded from 1321N1 cells expressing recombinant heteromultimeric rat P2X2/3 receptors. Other responses (fast DRG) were rapidly activating and desensitized almost completely during agonist application. These responses had properties similar to those recorded from 1321N1 cells expressing recombinant rat P2X3 receptors. A third group (mixed DRG) activated and desensitized rapidly (P2X3-like), but also had a slow, nondesensitizing component that functionally prolonged the current. Like the fast component, the slow component was activated by both ATP and alpha, beta-meATP and was blocked by the P2X antagonist TNP-ATP. But unlike the fast component, the slow component could follow high-frequency activation by agonist, and its amplitude was potentiated under acidic conditions. These characteristics most closely resemble those of rat P2X2/3 receptors. These data suggest that there are at least two populations of P2X receptors present on adult DRG nociceptive neurons, P2X3 and P2X2/3. These receptors are expressed either separately or together on individual neurons and may play a role in the processing of nociceptive information from the periphery to the spinal cord.

Animals↗

PubMed1999

Allosteric modulation and accelerated resensitization of human P2X(3) receptors by cibacron blue.

The activity of ATP as a fast neurotransmitter is mediated by the P2X family of ligand-gated ion channels. P2X receptor subtypes are subject to functional modulation by a diverse set of factors, including pH, divalent cations, and temperature. The human P2X(3) (hP2X(3)) receptor subunit is expressed primarily in sensory ganglia where it exists as either a homomultimeric receptor or, in combination with P2X(2), as a heteromultimeric receptor. This article describes the allosteric modulatory effect of the putative P2X receptor antagonist cibacron blue on the activity of recombinant hP2X(3) receptors. In 1321N1 cells expressing the hP2X(3) receptor, cibacron blue mediated a 3- to 7-fold increase in both the magnitude and the potency of ATP-activated Ca(2+) influx and transmembrane currents. The half-maximal concentration of cibacron blue required to mediate maximal potentiation (EC(50) = 1.4 microM) was independent of the agonist used to activate the hP2X(3) receptor. The nonselective P2 receptor antagonist PPADS (pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid) caused a rightward shift of the cibacron blue concentration-effect curve, whereas increasing concentrations of cibacron blue attenuated PPADS antagonism. In addition to potentiating the effects of ATP at the hP2X(3) receptor, cibacron blue also produced a 6-fold increase in the rate of hP2X(3) receptor recovery from desensitization (from T(1/2) = 15.9 to 2.6 min), as evidenced by its ability to restore ATP responsiveness to acutely desensitized receptors. Consistent with the properties of other ligand-gated ion channels, these results suggest that hP2X(3) receptor activity can be allosterically modulated by a ligand distinct from the endogenous agonist.

Adenosine Triphosphate↗

PubMed1998

Inhibition of adenosine kinase during oxygen-glucose deprivation in rat cortical neuronal cultures.

Adenosine kinase (AK) inhibitors potentiate the actions of endogenous adenosine (ADO) and ameliorate cerebral ischemic damage in animal models. The present study examined the effects of the AK inhibitor, 5-iodotubercidin (5-IT) in an in vitro model of neuronal ischemia, specifically, combined oxygen-glucose deprivation of rat cortical mixed neuronal-glial cultures. Oxygen-glucose deprivation caused extensive neuronal loss which was accompanied by a marked increase in ADO release into the extracellular medium, was ameliorated by exogenous ADO (10 microM(-1) mM), and was exacerbated by a high concentration of the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 10 microM). 5-IT (1 microM) had no effect on extracellular ADO levels nor on neuronal loss. However, AK activity in these cultures was markedly suppressed during oxygen-glucose deprivation. Taken together, these data demonstrate a marked down-regulation of AK activity during oxygen-glucose deprivation in this in vitro model, providing an endogenous mechanism contributing to the accumulation of extracellular ADO, which exerts neuroprotective effects by activating the ADO A1 receptor.

Adenosine↗

PubMed1998

Single and repeated episodes of ethanol withdrawal increase adenosine A1, but not A2A, receptor density in mouse brain.

A history of multiple ethanol withdrawal experiences has been shown to exacerbate the severity of future withdrawal episodes, and this sensitization of the withdrawal response has been hypothesized to represent a 'kindling' phenomenon. Since adenosine functions as an inhibitory modulator of seizure activity and may interact with ethanol to influence neuronal excitability, the present study was conducted to examine the effects of single and repeated episodes of ethanol withdrawal on adenosine A1 and A2A receptors in adult C3H/He mice. Mice were chronically exposed to ethanol vapor in inhalation chambers and tested for withdrawal seizures following multiple withdrawal (MW) experience (four cycles of 16 h ethanol intoxication interrupted by 8 h periods of abstinence), single withdrawal experience following 16 h (SW) or 64 h (CE) continuous ethanol intoxication, or no ethanol exposure (controls). Separate groups of mice from each withdrawal condition were used to generate pooled cortical and striatal tissue for ligand saturation experiments using [3H]cyclohexyladenosine to label A1 receptors and [3H]CGS 21680 to label A2A receptors. Results indicated that withdrawal seizures were significantly more severe in mice with multiple withdrawal experience in comparison to animals that experienced only a single withdrawal episode, even when total amount of ethanol exposure was equated among groups. The density of A1 receptors in cerebral cortex was significantly increased over controls 8 h following final ethanol withdrawal by approximately 35% in SW and CE groups, with the largest increase observed in the MW group (56%). Withdrawal treatment groups did not differ in cortical A1 binding sites immediately upon withdrawal from ethanol, and no significant differences in binding of [3H]CGS 21680 to striatal A2A receptors were observed following ethanol withdrawal. Ethanol exposure and withdrawal did not significantly alter ligand affinity for either adenosine receptor. These results indicate that adenosine A1 receptors are selectively upregulated during ethanol withdrawal and that the degree of upregulation may be enhanced following multiple withdrawal episodes. Further, these observations suggest that the upregulation of brain A1 receptors during ethanol withdrawal may represent a compensatory inhibitory response to increased seizure severity associated with repeated episodes of ethanol withdrawal.

Animals↗

PubMed1998

Adenosine kinase inhibitors.

Adenosine (ADO) is an endogenous modulator of intercellular signaling that provides homeostatic reductions in cell excitability during tissue stress and trauma. The inhibitory actions of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors regulating membrane cation flux, polarization, and the release of excitatory neurotransmitters. ADO kinase (AK; EC 2.7.1.20) is the key intracellular enzyme regulating intra- and extracellular ADO concentrations. Inhibition of AK produces marked increases in extracellular ADO levels that are localized to cells and tissues undergoing accelerated ADO release. Thus AK inhibition represents a mechanism to selectively enhance the protective actions of ADO during tissue trauma without producing the nonspecific effects associated with the systemic administration of ADO receptor agonists. During the last 10 years, specific inhibitors of AK based on the endogenous purine nucleoside substrate, ADO, have been developed. Potent AK inhibitors have recently been synthesized that demonstrate high specificity for this enzyme as compared to other ADO metabolic enzymes, transporters, and receptors. In both in vitro and in vivo models, AK inhibitors have been shown to potently increase ADO concentrations in a tissue and event specific fashion and to demonstrate potential clinical utility in animal models of epilepsy, ischemia, pain, and inflammation. AK inhibitors have demonstrated superior efficacy in these models as compared to other mechanisms of modulating ADO availability, and these agents exhibit reduced side-effect liabilities compared to direct acting ADO receptor agonists. The preclinical profile of AK inhibitors indicate that these agents may have therapeutic utility in a variety of central and peripheral diseases associated with cellular trauma and inflammation. Clinical trials are currently underway to evaluate the efficacy of AK inhibitors in seizure disorders and pain.

Adenosine↗

PubMed1997

[125I]BQ3020, a novel endothelin agonist selective for the endothelinB receptor subtype.

The endothelins represent a family of three (ET-1, ET-2, and ET-3) isopeptides that are derived from vascular endothelial cells and function as potent vasoconstrictors. Two endothelin receptor subtypes, ETA and ETB, have been cloned and are structurally similar to the large family of guanine nucleotide-binding protein (G-protein) linked receptors. The ET receptor subtypes can also be distinguished by their different pharmacological profiles.. A novel linear peptide fragment of endothelin-1 (ET-1), N-acetyl-[Ala11,15]ET-1[6-21] (BQ3020) has been identified as a potent and ETB-selective agonist. [125I]BQ3020 binds with high affinity (Kd = 31 pM) and limited capacity (Bmax = 570 fmol/mg protein) to a single class of recognition sites in rat cerebellum. High affinity [125I]BQ3020 binding is reduced in the presence of guanine nucleotides indicating an interaction with high and low affinity states of the ETB receptor. This protocol describes the use of [125I]BQ3020 as a specific radioligand for the ETB receptor.

Animals↗

PubMed1996

Chronic ethanol exposure selectively increases diazepam-insensitive [3H]RO15-4513 binding in mouse cerebellum.

The effect of chronic ethanol exposure and withdrawal on [3H]RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4] benzodiazepine-3-carboxylate) binding to diazepam-sensitive and diazepam-insensitive binding sites was determined in mouse brain. Neither chronic ethanol treatment nor withdrawal significantly altered total [3H]RO15-4513 binding in mouse cortex or cerebellum. However, diazepam-insensitive [3H]RO15-4513 binding density (Bmax) in cerebellum was significantly increased immediately following chronic ethanol treatment (60%) and at 8 h following withdrawal (75%). [3H]RO15-4513 binding affinity was not significantly influenced by chronic ethanol exposure or withdrawal. These results indicate that chronic ethanol treatment and withdrawal can selectively up-regulate diazepam-insensitive [3H]RO15-4513 binding sites and suggest that this unique GABAA receptor subtype may play some role in ethanol dependence and withdrawal.

Administration, Inhalation↗

PubMed1994

Pharmacological characterization of the rat cerebellar endothelin B (ETB) receptor using the novel agonist radioligand [125I]BQ3020.

A novel linear peptide fragment of endothelin-1 (ET-1), N-acetyl-[Ala11,15]ET-1[6-21] (BQ3020) has been identified as a potent and ETB-selective agonist. The present studies were conducted in order to characterize the binding of [125I]BQ3020 to the ETB receptor in rat cerebellum. [125I]BQ3020 (0.1 nM) bound with high specificity (90% of total binding) and selectivity for the ETB receptor. ET-1, ET-2, and ET-3 inhibited 0.1 nM [125I]BQ3020 binding with equivalent affinity (Ki values = 55-110 pM). The sarafotoxins S6a, S6b, and S6c also potently inhibited [125I]BQ3020 binding (Ki values = 55-2000 pM). The ETA-selective antagonist, BQ123 (100 microM) did not significantly inhibit [125I]BQ3020 binding. Ligand saturation studies indicated that [125I]BQ3020 labeled a single class of recognition sites with very high affinity (Kd = 31 pM) and limited capacity (Bmax = 570 fmol/mg protein). High affinity 0.1 nM [125I]BQ3020 binding was reduced by 40-50% in the presence of 1 mM guanine nucleotides. Additional competition studies indicated that ET-1 and ET-2 produced biphasic inhibition curves in competing for 0.5 nM [125I]BQ3020. The high affinity component of the ET-1 inhibition curve was subsequently eliminated in the presence of 1 mM GTP-gamma-S The guanine nucleotide sensitivity of [125I]BQ3020 binding offers the possibility that different functional consequences of ETB receptor activation may be mediated by multiple affinity states of the receptor. The present data demonstrate that [125I]BQ3020 is a potent and selective agonist for the ETB receptor that can discriminate high and low affinity states of the ETB receptor.

Animals↗

PubMed1994

Dithiothreitol, sodium chloride, and ethylenediaminetetraacetic acid increase the binding affinity of [125I]angiotensin IV to AT4 receptors in bovine adrenal cortex.

The present studies demonstrate that the sulfhydryl reducing agent, dithiothreitol (DTT), increases the specific binding of [125I]angiotensin IV ([125I]AIV) to AT4 receptors in bovine adrenal cortical membranes. Both the degree of stimulation and the pharmacological selectivity of [125I]AIV binding in the presence of DTT were quantitatively different depending on the contents of the assay buffer. Similar effects were also observed using a different sulfhydryl reducing agent, 2-mercaptoethanol (2-MCE). These sulfhydryl reducing agents (100 mM) produced a 200% increase in specific [125I]AIV binding in an assay buffer that has been used to characterize the novel AT4 receptor subtype. A much larger stimulation (700%) of specific [125I]AIV binding was found when the assay was conducted in a buffer that has been used to characterize ligand binding to the AT1 receptor. Ligand association studies indicated that 0.3 nM [125I]AIV displayed similar equilibrium kinetics and stability in both the AT4 and AT1 buffers. Ligand saturation studies indicated that [125I]AIV bound with high affinity (Kd = 6 nM) in the AT4 buffer system, but bound with lower affinity (Kd = 32 nM) in the AT1 buffer system. Removal of NaCl and EDTA from the AT4 buffer also resulted in low-affinity [125I]AIV binding (Kd = 33 nM). The subsequent inclusion of NaCl, EDTA, or DTT resulted in higher-affinity [125I]AIV binding (KdS = 3-14 nM). No significant effects on the apparent density (Bmax) of AT4 receptors were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗

PubMed1993

Characterization of the bisphosphonate recognition site on hydroxyapatite using radioligand binding techniques with [14C]citric acid.

The present studies characterize the binding of [14C]citric acid to synthetic hydroxyapatite (HA) crystals. [14C]Citric acid specifically bound to HA and was dependent upon the concentration of HA in the assay. The binding of [14C] citric acid to HA reached equilibrium within 20 min and remained stable for at least 90 min. Dissociation of bound [14C]citric acid was biphasic in nature since both rapid and more slowly reversible binding components were detected. Saturation experiments also indicated that [14C]citric acid labeled two recognition sites with different affinity (KdH = 42 nM and KdL = 24,000 nM) and density (BmaxH = 161 fmol/micrograms HA and BmaxL = 8.8 pmol/micrograms HA). Ligand competition experiments revealed that compounds that are known to readily bind bone (e.g., sodium pyrophosphate, methylene diphosphonic acid, etidronate) potently inhibited the binding of [14C]citric acid to HA, whereas compounds known to have poorer affinity for bone (e.g., oxalic acid and GABA) did not. Computer analysis of these inhibition curves revealed specific ligand interactions at two different affinity recognition sites. The present results indicate that [14C]citric acid binds discrete sites on synthetic HA in a fashion consistent with a specific labeling of the bisphosphonate recognition site. Analysis of the binding of [14C]citric acid to HA provides a useful method to further explore the structure activity relationships of novel compounds that have binding affinity for bone.

Binding Sites↗

PubMed1993

Adenosine agonists reduce conditioned avoidance responding in the rat.

Because adenosine agonists may possess therapeutic potential as antipsychotic agents, we examined the activity of several prototypic agents in vivo in blocking conditioned avoidance responding (CAR) in the rat, a behavioral test predictive of antipsychotic efficacy in humans. Potency in blocking CAR is directly proportional to potency in alleviating schizophrenia. Hence, the adenosine A1-selective agonists [cyclopentyl adenosine (CPA) and (R)-phenylisopropyl adenosine (R-PIA)], A2-selective agonists [CV-1808 and (2-(p-(carboxyethyl)-phenethylamino)-5'-N-ethyl-carboxamido adenosine (CGS 21680)], and a nonselective agonist [5'-N-carboxamido adenosine (NECA)] were examined in this test. Block of CAR was first determined for standard antipsychotic agents [ED50 mg/kg, IP, and 95% confidence level (CL) in parentheses], such as haloperidol [0.23 (0.18, 0.39)], trifluoroperazine [(0.9 (0.7, 1.0)], thioridazine [12.5 (10.5, 15.3)], metoclopramide [7.8 (6.4, 9.2)], and chlorpromazine [4.9 (4.2, 5.9)]. The paradigm consisted of a light- and tone-signaled footshock that could be avoided via a discrete lever press. Affinity for A1 and A2 binding sites in brain tissue from Fischer 344 rats was ascertained to be similar to that seen in other rodent strains. Each adenosine agonist blocked CAR. NECA [ED50 value (95% CL) = 0.07 (0.004, 0.12) mg/kg, IP] was the most potent agent, followed by: R-PIA [0.34 (0.23, 0.44)]; CGS 21680 [1.1 (0.8, 2.0)]; CV-1808 [1.3 (1.0, 1.8)]; and CPA [1.5 (1.3, 1.7)]. Pretreatment with caffeine (25 mg/kg, IP, -10 min) blocked the inhibition of CAR produced by the adenosine agonists, suggesting the event is mediated via purinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗