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K M Standifer

Publications and source records attributed to K M Standifer.

30 records · Page 2Linked to original sources
PubMed1994

Biochemical and pharmacological characterization of mu, delta and kappa 3 opioid receptors expressed in BE(2)-C neuroblastoma cells.

Total opioid binding in the human neuroblastoma cell line BE(2)-C has a density similar to that found in brain, with a Bmax value of 383 +/- 60 fmol/mg protein and a KD of 0.4 +/- 0.07 nM for the nonselective opioid antagonist 3H-diprenorphine. Selective assays reveal a binding distribution of mu (38%), delta (16%) and kappa 3 (43%) opioid receptors. There is no observable kappa 1 or kappa 2 binding. The sum of the Bmax values in the selective binding assays (370 +/- 39 fmol/mg protein) approximates closely that observed with 3H-diprenorphine, suggesting that mu, delta and kappa 3 sites account for most of the binding. The binding selectivities of various opiates and opioid peptides in the BE(2)-C cells are similar to those in rat brain. Delta and mu binding are defined easily by traditional selective ligands. The binding profiles also distinguish clearly mu from kappa 3 binding. The selective mu ligand DAMGO competes with mu binding over 35-fold more potently than kappa 3 binding, whereas morphine shows a 10-fold selectivity. Functionally, selective mu, delta and kappa 3 agonists inhibit forskolin-stimulated cAMP accumulation through distinct receptor mechanisms that are pertussis toxin-sensitive. In addition to demonstrating that BE(2)-C cells provide a useful model system for studying mu, kappa 3 and delta receptors, these studies confirm that kappa 3 receptors represent a pharmacologically distinct receptor class in this cell line.

Animals↗
PubMed1994

Opioid binding in giant toad and goldfish brain.

Opiate receptor expression in phylogenetically different species has played an important role in the study of opioid receptor pharmacology. Total opioid binding measured with the nonselective ligand 3H-diprenorphine reveals a Bmax of 21.7 +/- 1.37 fmol/mg tissue wet wt and a KD of 0.17 +/- 0.03 nM in Bufo marinus (giant toad), as well as a Bmax of 18.17 + 0.41 fmol/mg tissue wet wt and a KD of 0.47 +/- 0.18 nM in Carassius auratus (goldfish). Despite the similar levels of 3H-diprenorphine binding, the composition of binding subtypes in the two species differs. Approximately 30% of total binding corresponds to mu receptors in both species, whereas neither kappa 1 nor delta binding can be detected. However, the remaining 70% of binding differs between the toad and goldfish. In the toad, the non-mu binding corresponds to kappa 2 sites, whereas in the goldfish, the non-mu binding corresponds to kappa 3 sites. The sites can be distinguished biochemically, as well as pharmacologically. After affinity labeling the sites with 3H-NalBzoH, the retention times on an ion-exchange column differ for the peaks of kappa binding in the two species. Although Bufo marinus (giant toad) and Carassius auratus (goldfish) brains express kappa and mu opioid binding, the kappa subtypes in these two species differ.

Animals↗
PubMed1993

Modulation of Mu1 opioid binding by magnesium: evidence for multiple receptor conformations.

Previous studies have revealed that MgSO4 markedly increases mu1 binding and is associated with approximately a 10-fold slower dissociation rate, implying an increase in receptor affinity. Yet, saturation studies showed only a slight change in Kd values with a significant increase in the Bmax values. We now extend these observations using detailed kinetic approaches to better understand the role of MgSO4 on mu1 binding. Using blocking concentrations of the delta-selective ligand DPDPE, low concentrations of [3H]DADLE label mu1 sites almost exclusively in calf thalamic homogenates in the presence of either MgSO4 or EDTA, as demonstrated in competition studies. Saturation studies performed under equilibrium conditions with EDTA or MgSO4 reveal Bmax values of 1.89 +/- 0.20 and 4.4 +/- 0.19 fmol/mg wet weight tissue, respectively, with Kd values of 3.8 +/- 0.8 and 1.92 +/- 0.27 nM. Despite the small but significant 2-fold difference in Kd values under equilibrium conditions, kinetic studies reveal the dissociation rate constants (k-1) for the two assay conditions vary by almost 8-fold. NaCl increases the dissociation rate far more in assays performed in EDTA, while binding in the presence of MgSO4 is more sensitive to the stabilized GTP analog 5'-guanylylimidodiphosphate. Association studies also reveal a 20-fold difference between rate constants (k+1). The apparent association rate constants (kobs) in the presence of EDTA are concentration-dependent, whereas varying the concentration of [3H]DADLE has no effect on the rate constants under MgSO4 conditions. Our data suggest a model in which mu1 binding comprises two sequential steps and at least three distinct receptor binding states.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Affinity labeling of mu and kappa receptors with naloxone benzoylhydrazone.

Naloxone benzoylhydrazone (NalBzoH) labels both mu and kappa receptors in standard homogenate binding assays. We now report that [3H]NalBzoH can effectively photoaffinity label opioid receptors. By modifying [3H]NalBzoH binding conditions, we can selectively label either mu or kappa 3 receptors in calf striatal membranes or classical U50,488-sensitive kappa 1 receptors in guinea pig cerebellar membranes. After removal of unbound radioligand, the [3H]NalBzoH-labeled membranes were irradiated with UV light to couple the bound radioligand to its binding site. No specific mu, kappa 1, or kappa 3 binding remained after a 20-hr dissociation at 25 degrees without UV irradiation. In contrast, approximately 45% of mu and 40% of kappa 1 and kappa 3 binding remained after 2 min of UV exposure. In time course studies, increasing the UV exposure from 30 sec to 3 min produced a progressive increase in radioligand incorporation, which did not increase further with UV exposure times up to 5 min. A portion of the binding in irradiated membranes appeared to be covalently coupled to proteins. Following solubilization of irradiated membranes with sodium dodecyl sulfate, approximately 30-40% of the specifically bound radioactivity precipitated with trichloroacetic acid (TCA). These levels of TCA-precipitable binding corresponded to the amount of dissociation-resistant binding described above. No specific binding could be TCA precipitated in samples that were not exposed to UV. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]NalBzoH-labeled membranes revealed a number of labeled peaks with levels of radioactivity that did not correspond to the intensity of the protein bands, suggesting that this technique was not randomly labeling proteins. This approach may be a useful method to affinity label, characterize, and purify mu and kappa opiate receptor subtypes.

Affinity Labels↗
PubMed1990

Reduction by central beta-funaltrexamine of food intake in rats under freely-feeding, deprivation and glucoprivic conditions.

The present study evaluated the central effects of beta-funaltrexamine (B-FNA), a non-equilibrium antagonist of mu-opioid receptors and a reversible agonist of kappa-opioid receptors upon food intake in rats under freely-feeding, deprivation and glucoprivic conditions. B-FNA elicited distinct short-term and long-term actions, consistent with binding studies demonstrating its reversible kappa agonist actions and its irreversible mu receptor blockade. Whereas B-FNA (1-20 micrograms, i.c.v.) significantly stimulated free feeding for up to 6 h, B-FNA (10-20 micrograms) significantly inhibited (35-41%) free feeding at 24, 48 and 72 h after injection, a pattern temporally similar to its biochemical opioid effects. Pretreatment (24 h) with B-FNA (10-20 micrograms) significantly inhibited (33-49%) the increased intake following 24 h of food deprivation. Pretreatment (24 h) with B-FNA (10-20 micrograms) also significantly inhibited (75-100%) the increased glucoprivic intake induced by 2-deoxy-D-glucose. The short-term stimulation of food intake by central B-FNA was antagonized by the selective kappa antagonist, nor-binaltorphamine, but was unaffected by pretreatment 24 h earlier with the mu antagonist, B-FNA. Significant reductions in striatal (89%) and hypothalamic (46%) mu-opioid binding occurred in rats pretreated (24 h) with B-FNA; the low levels of delta binding in these structures precluded interpretation of B-FNA effects. These data indicate the importance of the mu-opioid receptor in the modulation of different forms of feeding behavior, and underscores the ability of selective opioid antagonists to delineate precise functional roles for different opioid receptor subtypes.

Animals↗
PubMed1989

Pharmacological characterization of morphine-6 beta-glucuronide, a very potent morphine metabolite.

Morphine-6 beta-glucuronide is a major metabolite of morphine with potent analgesic actions. To define more fully the importance of this compound in morphine action, we have compared the analgesic actions of morphine and its 6 beta-glucuronide metabolite after both peripheral and central administration. Given s.c., morphine-6 beta-glucuronide elicited analgesia with an effect approximately twice that of morphine due, in part, to its long duration of action and also inhibited gastrointestinal motility. Both actions were easily reversed by naloxone (s.c.). However, when injected either i.c.v. or intrathecally, morphine-6 beta-glucuronide was approximately 90- and 650-fold more potent an analgesic than morphine, respectively. Whereas morphine in these studies was equipotent at both levels of the neuraxis as an analgesic, the 6 beta-glucuronide was approximately 5-fold more effective at the level of the spinal cord than supraspinally. The mu 1-selective antagonist naloxonazine blocked the analgesic effect of systemic and i.c.v. morphine-6 beta-glucuronide much as it blocked morphine, implying a role for mu1 receptors in these actions. Like morphine, morphine-6 beta-glucuronide analgesia after intrathecal injection was not sensitive to naloxonazine, suggesting a mu2 mechanism within the spinal cord. Together, these results imply that morphine-6 beta-glucuronide elicited its analgesic actions through the same receptor mechanisms as morphine. Mice highly tolerant to morphine after implantation of morphine pellets showed cross-tolerance to morphine-6 beta-glucuronide (s.c.). The high potency of morphine-6 beta-glucuronide strongly suggests that this metabolite plays an important role in morphine's actions.

Analgesia↗
PubMed1988

Irreversible binding and recovery of the norepinephrine uptake system using an alkylating derivative of norepinephrine.

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.

Adrenergic beta-Antagonists↗
PubMed1987

Carbostyril derivatives having potent beta-adrenergic agonist properties.

Derivatives carrying a substituent in the para position of the phenyl group of 8-hydroxy-5-[2-[(1-phenyl-2-methylprop-2-yl)amino]-1-hydroxyethyl] carbostyril (10) were prepared and their effects on beta-adrenoceptors evaluated in vitro. Unsubstituted compound 10, iodo 11, amino 12, and bromoacetamido 13 derivatives (all racemic) bound to the receptor with 15-100-fold lower dissociation constants than that of (-)-isoproterenol. All the above compounds stimulated adenylate cyclase more potently than (-)-isoproterenol. The intrinsic activities of compounds 10 and 12 were equal to that of (-)-isoproterenol. The intrinsic activities of compounds 11 and 13 were 1.3 and 1.2 times that of (-)-isoproterenol, respectively. Treatment of membrane preparations with bromoacetamido derivative 13 resulted in an irreversible loss of binding sites, and thus, 13 seems to be an alkylating affinity label for beta-adrenoceptors.

Adenylyl Cyclases↗
PubMedDate not supplied

Carbostyril-based beta-adrenergic agonists: evidence for long lasting or apparent irreversible receptor binding and activation of adenylate cyclase activity in vitro.

The interaction of the carbostyril derivatives 5-[2-[[1-(4-aminophenyl)-2-methyl-prop-2-yl]amino]-1-hydroxyethyl]- 8-hydroxycarbostyril (carbo-amine) and 5-[2-[[3-[4-(bromoacetamido)phenyl]-2-methylprop-2-yl]amino]-1- hydroxyethyl]-8-hydroxycarbostyril (carbo-Br) with the rat reticulocyte beta-adrenoreceptor system has been partially characterized. In the absence of a guanine nucleotide, the concentration of carbo-amine, carbo-Br and (-)isoprenaline that inhibited (-)-[125I]iodocyanopindolol ([125I]CYP) binding by 50% (IC50) was 5.9 +/- 0.2, 3.3 +/- 0.3 and 49 +/- 3 nM, respectively. In the presence of a guanine nucleotide, the IC50 values were carbo-amine, 21 +/- 0.6 nM; carbo-Br, 7.6 +/- 0.3 nM and (-)isoprenaline, 813 +/- 66 nM. Preincubation of membranes with either of the carbostyril congeners followed by washing reduced specific [125I]CYP binding capacity without changing the KD value for the remaining receptors. The beta-antagonist nadolol largely prevented the receptor reduction induced by the carbostyril compounds. Incubation of membranes for 18 h at 25 degrees C resulted in an 11% recovery of the carbo-amine-induced receptor loss and no recovery of the receptors lost by preincubation with carbo-Br. However, the carbo-amine induced receptor loss could be largely reversed (80%) by membrane heating at 45 degrees C whereas little reversal (less than 10%) was observed with the carbo-Br pretreated membranes. The concentration of carbo-amine, carbo-Br and (-)isoprenaline that stimulated half-maximal cAMP formation in reticulocyte membranes was 17.8 +/- 3.1, 8.2 +/- 2.1 and 241 +/- 17 nM, respectively, and all 3 agonists produced the same maximal response. Initial cAMP formation stimulated by the carbostyril derivatives and (-)isoprenaline was blocked by concurrent addition of propranolol after 7 min of incubation with either of the two carbostyril derivatives did not affect further cAMP production whereas with (-)isoprenaline further cAMP production was blocked.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMedDate not supplied

G proteins and opioid receptor-mediated signalling.

Most opioid receptor-mediated functions appear to be mediated through G protein interactions, therefore an understanding of opioid signalling requires knowledge of those interactions. This review chronicles the studies examining these interactions for all the opioid receptor subtypes, both in vivo and in vitro.

Animals↗
PubMedDate not supplied

GABA(A) receptor binding and localization in the tiger salamander retina.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the retina and also appears to act as a trophic factor regulating photoreceptor development and regeneration. Although the tiger salamander is a major model system for the study of retinal circuitry and regeneration, our understanding of GABA receptors in this species is almost exclusively based on the results of physiological studies. Therefore, we have examined the pharmacological binding properties of GABA(A) receptors and their anatomical localization in the tiger salamander retina. Radioligand-binding studies showed that specific 3H-GABA binding to GABA(A) receptors was dominated by a single high-affinity binding site (Kd = 15.6+/-6.9 nM). Specific binding of 3H-GABA was almost completely eliminated by muscimol (Ki = 105+/-62 nM) and bicuculline (Ki = 14.3+/-2.2 microM); however, SR-95531 only displaced about 40% of specific 3H-GABA binding (Ki = 35.0+/-3.8 nM). These data indicate that there are at least two subtypes of GABA(A) receptors present in the salamander retina that can be distinguished by their antagonist binding properties: one sensitive to both bicuculline and SR-95531, and one sensitive to bicuculline but insensitive to SR-95531. Because localization of GABA receptors in the salamander retina by immunocytochemistry is problematic, GABA(A) receptors were localized by fluorescent ligand binding combined with immunocytochemical labeling for cell specific markers. Binding of fluorescently labeled muscimol to GABA(A) receptors was present in both plexiform layers and on photoreceptor cell bodies. GABA(A) receptors in the outer plexiform layer were localized to both photoreceptor terminals and horizontal cell processes.

Ambystoma↗