(+)-[3H]SKF 10,047, (+)-[3H]ethylketocyclazocine, mu, kappa, delta and phencyclidine binding sites in guinea pig brain membranes

Binding of the opiates (+)-[3H]SKF 10,047 [N-allylnormetazocine; (+)-[3H]SKF] and (+)-[3H]ethylketocyclazocine [(+)-[3H]EKC] were compared to mu, kappa and delta and phencyclidine (PCP) receptor binding in guinea pig brain membranes. (+)-[3H]SKF and (+)-[3H]EKC binding were not blocked by naloxone, and had different drug selectivity compared to mu, kappa and delta binding sites. The number of binding sites, drug selectivity and region distribution in brain were similar for (+)-[3H]SKF and (+)-[3H]EKC. Sigma opiates that are associated with psychotomimetic activities, such as pentazocine, cyclazocine, SKF 10,047 and bremazocine, were potent inhibitors of (+)-[3H]SKF and (+)-[3H]EKC binding. Haloperidol was the most potent inhibitor of (+)-[3H]SKF binding. Haloperidol and sigma opiates demonstrated biphasic displacement of [3H]PCP binding, suggesting that [3H]PCP labelled two sites. PCP had a similar affinity for both (+)-[3H]SKF and [3H]PCP binding sites in the presence of 100 mM NaCl. The highest concentrations of (+)-[3H]SKF and (+)-[3H]EKC bindings sites were in the hypothalamus, anterior pituitary, midbrain, pons and medulla.     

Unusual binding of [3H] MDL 72222 in guinea pig hippocampus

[³H] MDL 72222 labeled a non-homogeneous population of sites in guinea pig hippocampal membranes (K_d1 = 1 nM; K_d2 = 60 nM). The binding was not sodium dependent. Competition studies with a variety of characterizing agents showed displacement of [³H] MDL 72222 binding by 5-HT uptake inhibitors. [³H] MDL 72222 binding was not effectively displaced by established 5-HT₃ antagonists. MDL 72222, fluoxetine, fluvoxamine and citalopram competitively inhibited the uptake of [³H] 5-HT into guinea pig hippocampal synaptosomes with K_i values of 1.97, 0.02, 0.023, 0.049 μM, respectively. The results demonstrate that [³H] MDL 72222 labels a non-homogeneous population of sites in guinea pig brain, as well as inhibiting 5-HT uptake into synaptosomal preparations.     

Localisation of [3H]clonidine binding to membranes from guinea pig renal tubules

The selective radioligand [3H]clonidine has been used to localise alhpa 2 adrenoceptors in guinea pig kidney. Chemical sympathectomy with 6-hydroxydopamine produced no significant change in the number of sites labeled by [3H]clonidine indicating that the majority of binding sites were not located on sympathetic nerve terminals. Binding was enhanced in membranes prepared from renal tubules and considerably reduced in preparations from glomeruli. Subcellular fractions of renal cortex revealed that binding was to plasma membranes and that the greatest binding capacity was present in the fraction rich in basal lateral membranes. It is concluded that the major concentration of renal alpha 2 adrenoceptors are present on renal tubules and that they may be localised to a particular pole of the renal tubule cell.     

(+)-[3H]-PN 200-110 binding to cell membranes and intact strips of portal vein smooth muscle: characterization and modulation by membrane potential and divalent cations.

1. Specific binding of the calcium-antagonist dihydropyridine derivative, (+)-[3H]-PN 200-110 (isradipine), to cell membranes of equine portal vein smooth muscle was compared with binding to intact strips isolated from rat portal veins. 2. Specific binding to vascular smooth muscle membranes was of high affinity, saturable and reversible. The dissociation constant obtained from association and dissociation kinetics of (+)-[3H]-PN 200-110 was similar to that obtained from equilibrium binding and competition experiments. 3. Specific binding of (+)-[3H]-PN 200-110 was completely displaced by unlabelled dihydropyridines. Among other calcium antagonists, D888 and (+)-cis-diltiazem partially inhibited the binding at 25 degrees C. At 37 degrees C, only (+)-cis-diltiazem stimulated the binding. LaCl3, CdCl2, NiCl2, CoCl2 had inhibitory effects, whereas KCl and NaCl had no effect. 4. When intact strips of portal vein were incubated in high external potassium concentrations for 30 min, the Kd was lowered to 0.04 +/- 0.01 nM from the control value of 0.14 +/- 0.02 nM (n = 5), thereby indicating that (+)-[3H]-PN 200-110 bound to voltage-dependent calcium channels, with a higher affinity, in the depolarized state. 5. When external Ca2+ was removed or substituted with Ba2+ or Sr2+, Kd values increased suggesting that the dihydropyridine binding to intact strips was modulated by binding of Ca2+ ions to voltage-dependent calcium channels.     

3H]DTG and [3H](+)-3-PPP label pharmacologically distinct sigma binding sites in guinea pig brain membranes.

The interaction of various compounds with sigma binding sites was examined in membranes prepared from whole guinea pig brain. Whereas [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine labeled a single population of binding sites exhibiting a Kd of 43 nM, [3H]1,3-di-o-tolylguanidine bound to two sites having Kds of 35 and 212 nM, and to a greater maximum number of sites than [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine. Haloperidol, 1,3-di-o-tolylguanidine, BMY 14802, and (-)-pentazocine each displayed nearly equal affinity for binding sites labeled by [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [3H]1,3-di-o-tolylguanidine, whereas (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine was 3 times more potent in inhibiting [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine than [3H]1,3-di-o-tolylguanidine binding. In contrast, (+)-SKF 10,047, (+)-cyclazocine and (+)-pentazocine exhibited more than 9-fold higher affinity for [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine than [3H]1,3-di-o-tolylguanidine binding sites. Dextromethorphan was 15-fold more potent against [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine than [3H]1,3-di-o-tolylguanidine, inhibited [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding in a biphasic manner, and inhibited [3H]haloperidol and [3H](+)-SKF 10,047 binding with potencies similar to those obtained against [3H]1,3-di-o-tolylguanidine and [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, respectively. Phenytoin increased [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [3H](+)-SKF 10,047 binding, but did not enhance [3H]1,3-di-o-tolylguanidine or [3H]haloperidol binding. However, the potency of dextromethorphan to inhibit [3H]1,3-di-o-tolylguanidine binding was increased in the presence of phenytoin.(ABSTRACT TRUNCATED AT 250 WORDS)     

(+)-[3H]SK&F 10,047 binding sites in rat liver

Stereospecific binding sites for (+)-[3H]SKF 10,047 have been demonstrated in rat liver. These binding sites were shown to be opioid receptors of the sigma type, and this was confirmed by their binding properties: reversibility, saturability, stereospecificity, effects of ions and various pharmacological drugs on (+)-[3H]SK sigma F 10,047 binding. The authors suggest that (+)-[3H]SKF 10,047 binding sites in liver and brain are a part of novel 'sigmergic' regulatory system.     

Pharmacologic characterization of [3H]dopamine and [3H]spiperone binding in mouse cerebellum

• 1.1. [³H]Dopamine and [³H]spiperone binding to cerebellar homogenates was characterized utilizing dopaminergic agonists, antagonists and non-dopaminergic drugs.
• 2.2. The [³H]DA binding to low affinity binding sites reveals a heterogenous population consisting of dopaminergic as well as serotonergic and noradrenergic sites. However, the high affinity binding of [³H]DA reflects dopaminergic sites, although a small contribution of serotonergic and noradrenergic binding sites cannot be excluded.
• 3.3. [³H]Spiperone also labels a heterogenous population of binding sites which, however, are mainly dopaminergic.

     

Interactions of spironolactone with (+)-[3H]-isradipine and (-)-[3H]-desmethoxyverapamil binding sites in vascular smooth muscle.

Spironolactone partially inhibited the specific binding of (+)-[3H]-isradipine and (-)-[3H]-desmethoxyverapamil to vascular smooth muscle membranes. It is suggested that spironolactone interacts at a binding site of the calcium channel complex and allosterically modulates ligand binding at receptor sites in the channel.     

Inhibition of [3H]kainic acid receptor binding by divalent cations correlates with ion affinity for the calcium channel

Since the neurotoxicity of kainic acid may be due to the opening of membrane channels for calcium ions for (Ca2+), the effects of Ca2+ and other cations were examined on the specific binding of [3H]kainic acid to membranes from the forebrain of the rat. [3H]Kainic acid bound to a high affinity site (KD = 5.6 nM) that was inhibited in a concentration-dependent manner by Ca2+ ions with an IC50 of 3.2 mM. In the presence of 1 mM Ca2+, the KD of the binding of [3K]kainic acid increased to 11.1 nM without any change in the Bmax. The divalent cations, manganese and cobalt, also were potent inhibitors of the binding of [3H]kainic acid, while barium and strontium were much weaker. The inhibitory effects of Ca2+ on the binding of [3H]kainic acid were blocked by the inorganic Ca2+ channel blockers, cadmium and lanthanum. These data suggest that Ca2+ modulates the binding affinity [3H]kainic acid through an allosteric interaction between the binding site on the Ca2+ channel and the kainic acid receptor.     

Consequences of specific [3H]ouabain binding to guinea pig left atria and cardiac cell membranes

An analysis of [3H]ouabain binding to electrically stimulated, contracting guinea pig left atria gave the following results. (1) A non-linear Scatchard plot with at least two binding sites: a high-affinity site (KD 1.1 X 10(-6) M) with about 430 receptors/micron2 related to positive inotropy, and a low-affinity site (KD' 2.1 X 10(-4) M) with about 18,000 receptors/micron2, possibly related to (Na+ + K+)ATPase inhibition. A crude left atrial homogenate gave about 530 receptors/micron2. (2) Half-maximal positive inotropic effects occurred at about 4 X 10(-7) M. (3) 86Rb+-uptake was significantly increased at all inotropic ouabain concentrations (10(-7) - 10(-6) M). Toxic concentrations (above 2 X 10(-6) M) inhibited 86Rb+-uptake (half-maximal inhibition at about 5 X 10(-6) M). [3H]Ouabain binding to partly purified guinea pig cardiac cell membranes showed: (a) linear Scatchard plots for (Mg2+, Pi)- and (Na+, ATP, Mg2+)-supported binding (KD 1.18 X 10(-7) M and 1.49 X 10(-7) M, respectively); (b) non-linear Scatchard plots for (Tyrode + ATP)-supported binding (KD 4.7 X 10(-7) M; KD' 6 X 10(-6) M); and (c) half-maximal [3H]ouabain binding occurred at a lower concentration (about 3.2 X 10(-7) M) than half-maximal inhibition of (Na+ + K+)ATPase activity (about 7.2 X 10(-7) M). Thus, we conclude that there may be more than one type of ouabain binding site in guinea pig left atria, and that measurable inhibition of (Na+ + K+)ATPase is not necessarily related to positive inotropy in the guinea pig.     

Effects of pentazocine and other opioids on the potassium-evoked release of [3H]noradrenaline from guinea pig cortical slices

Noradrenaline release and its modulation via presynaptic opioid receptors were examined in guinea pig cortical slices. Slices preloaded with [3H]noradrenaline were superfused in the presence of desipramine (1 microM) and were stimulated by 16 mM K+. 1-Pentazocine inhibited the K+-evoked release of [3H]noradrenaline in a dose-dependent manner (3 x 10(-7)-10(-5) M), while d-pentazocine did not inhibit. This inhibitory effect of 1-pentazocine was antagonized by Mr 2266 (10(-6) M) but not by naloxone (10(-6) M). Among other opioids, dynorphin A-(1-13) and ethylketocyclazocine (kappa agonists) decreased the K+-evoked release of [3H]noradrenaline. Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO, mu agonist) also inhibited [3H]noradrenaline release but was less potent than the kappa agonists. [D-Pen2,D-Pen5]enkephalin (DPDPE, delta agonist) and phencyclidine (sigma agonist) had no effects on the stimulated release of [3H]noradrenaline. Thus, it was shown that kappa receptors are the major subtype of opioid receptor involved in modulation of noradrenaline release in guinea pig cortex, and that 1-pentazocine inhibits the K+-evoked release of noradrenaline through activation of these receptors.     

Effects of four diltiazem stereoisomers on binding of d-cis-[3H]diltiazem and (+)-[3H]PN200-110 to rabbit T-tubule calcium channels.

Diltiazem, a benzothiazepine Ca2+ antagonist, has four stereoisomeric forms: d- and l-isomers of the cis and trans forms. All four isomers were shown to completely inhibit the binding of d-cis-[3H]diltiazem to rabbit T-tubule Ca2+ channels. The potency of the inhibition was in the order: d-cis greater than l-cis greater than d-trans = l-trans. The Hill slope for each inhibition was close to unity. The l-cis, d-trans and l-trans isomers had no effect on dissociation of the d-cis[3H]diltiazem-benzothiazepine receptor complex. These results indicate that all four isomers bind to benzothiazepine receptors. Furthermore, all of the isomers modulated (+)-[3H]PN200-110 binding. At 37 degrees C, only the d-cis isomer stimulated the binding, whereas the others showed inhibition. At 2 degrees C, all of them inhibited the binding. Both trans isomers exerted virtually the same, weak effects on the binding. It is concluded that the effects of diltiazem on radioligand binding to Ca2+ channels are highly stereospecific for the d-cis isomer.     

Subcellular distribution of (+)-[3H]SKF 10,047 binding sites in rat liver.

The distribution of (+)-[3H]SKF 10,047 binding sites and the distribution of the established plasma membrane, nuclear, mitochondrial and endoplasmic reticulum markers in subcellular fractions of rat liver have been studied. The distribution profile of (+)-[3H]SKF 10,047 binding sites coincided with that of NADPH-cytochrome c reductase, the endoplasmic reticulum marker. (+)-[3H]SKF 10,047 binding sites in rat liver are therefore suggested to be located on the endoplasmic reticulum membrane and to represent a membrane-bound enzyme.     

Modulation of [3H]flunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine

The influence of phosphatidylserine on ligand binding to the benzodiazepine/GABA receptor complex was assessed in rat cerebellar synaptic membranes and in a detergent-solubilized membrane preparation. Intact synaptic membranes or membranes solubilized with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate) were incubated with a range of concentrations of phosphatidylserine for 2 h at 4 degrees C, prior to use in radioligand binding assays. Phosphatidylserine, an endogenous membrane phospholipid, facilitated the site-specific binding of [3H]flunitrazepam to synaptic membranes and CHAPS-solubilized preparations. In addition, phosphatidylserine inhibited the facilitation of [3H]flunitrazepam binding induced by either cartazolate or gamma-aminobutyric acid (GABA). Although the maximum effect (38% facilitation of [3H]flunitrazepam binding; greater than 90% inhibition of the cartazolate action) was produced using 130 microM phosphatidylserine, a significant enhancement of [3H]flunitrazepam binding could be observed upon preincubation of synaptic membranes with concentrations of phosphatidylserine as low as 5 microM. These results suggest that endogenous phosphatidylserine may play a role in the regulation of benzodiazepine/GABA receptor function, possibly through modulation of the mechanisms which functionally link the various components of this complex receptor system.     

Modulation of affinity and density of LTB4 receptors on guinea pig lung membranes by divalent cations and guanine nucleotides.

We investigated the mechanism by which guanine nucleotides and divalent cations modulate the affinity and apparent density of high-affinity receptors for Leukotriene B4 (LTB4) on guinea pig lung membranes (GPLM). Divalent cations (Mg2+ = Ca2+ greater than Mn2+) stimulated, whereas EDTA inhibited (IC50 = 0.31 +/- 0.08 mM) binding of [3H]LTB4. Saturation analysis demonstrated that omission of divalent cations caused a two-fold reduction in apparent site density, (B max = 297 +/- 24 fmol/mg protein vs. 149 +/- 21 fmol/mg protein, P less than 0.01, for control and EDTA-treated respectively), but no significant change in receptor affinity (KD = 0.67 +/- 0.16 nM and 1.01 +/- 0.19 nM, P greater than 0.05). Competition experiments with LTB4 and the low-affinity (Ki = 165 nM) competitive LTB4-antagonist U75302, also demonstrated that EDTA caused a significant reduction (1.7 and 3.6-fold, P less than 0.05 and P less than 0.01, respectively), in affinity to both ligands. In the same experiments, the the guanine nucleotide analog GppNHp also reduced the affinity for LTB4 and U75302, similar to that observed with EDTA, suggesting that removal (Mg2+), or addition (GppNHp), of allosteric modulators of G-protein(s), causes reduction in receptor affinity. Saturation experiments also demonstrated that GppNHp, or GTP(gamma S), caused a significant reduction (40-50%) in receptor density. A larger reduction in affinity for U75302 (3- to 3.6-fold) than for LTB4 (1.7-fold) was induced by EDTA as well as GTP analogs.(ABSTRACT TRUNCATED AT 250 WORDS)