Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Decreased [3H]nitrendipine binding in the brainstem of deoxycorticosterone-NaCl hypertensive rats

Binding studies with the 1,4-dihydropyridine calcium channel antagonist [3H]nitrendipine [( 3H]NTD) were performed in uninephrectomized, deoxycorticosterone (DOCA)-NaCl hypertensive rats and vehicle treated normotensive control littermates. After 6 weeks of treatment, hypertensive (199 mmHg, systolic arterial pressure) DOCA rats showed significantly increased heart, left ventricle, and kidney weight in contrast to normotensive (135 mmHg) controls. [3H]NTD binding in the brainstem was significantly reduced (51 +/- 5 fmol/mg protein) in DOCA-NaCl rats, as compared to controls (116 +/- 24 fmol/mg protein). However, no significant differences were found in the [3H]NTD dissociation constants for DOCA-NaCl (0.43 +/- 0.03 nM) or control rats (0.62 +/- 0.06 nM). Cerebral cortical and left ventricular tissue showed no significant alterations in receptor binding density or affinity. Specific [3H]NTD binding was not significantly altered in other selected brain regions or the atria. These data suggest that alterations in the dihydropyridine binding sites associated with calcium channels in the brainstem may be involved in the etiology of DOCA-NaCl-induced hypertension.     

Decreased density of [3H]TCP binding following antipsychotic drug withdrawal in rats

Antipsychotic drugs have been reported to increase the expression of subunits of the NMDA receptor at the level of mRNA but it is not clear whether such effects are apparent at the level of the radioligand binding or receptor protein. Therefore, we examined the effect of treatment of, and withdrawal from, haloperidol, chlorpromazine, olanzapine or clozapine on the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP ) to the open ion channel of the NMDA receptor in rat caudate-putamen, hippocampus and frontal cortex. [3H]TCP binding was not significantly different in the caudate-putamen, hippocampus and cortex after three months of treatment with any antipsychotic drug. There were significant decreases in [3H]TCP binding in rat caudate-putamen and cortex, but not hippocampus, one month after ceasing treatment. Decreases in the caudate-putamen were detected in rats previously treated with chlorpromazine (0.1 mg/kg/day) and clozapine (0.1 and 1.0 mg/kg/day). In the cortex, decreases in [3H]TCP binding were also detected in rats previously treated with olanzapine (0.1 mg/kg/day) for three months. These data suggest that changes in the NMDA receptor associated ion channels occur following antipsychotic drug withdrawal.     

Characterization of [3H]cholecystokinin octapeptide binding to mouse brain synaptosomes: Effects of neuroleptics

The presence of high concentrations of both dopamine and cholecystokinin (CCK) in the striatum and in various limbic structures suggests that the CCK may not only influence dopaminergic transmission, but it also may be relevant to the psychopathology of schizophrenia and to the therapeutic effects of neuroleptics. By using a synaptosomal fraction isolated from the mouse cerebral cortex and [propionyl-³H]CCK8-sulphate ([³H]CCK8S) as a ligand, a single binding site for [³H]CCK8 with aK _d value of 1.04 nM and aB _max value of 42.9 fmol/mg protein was identified. The competitive inhibition of [³H]CCK8S binding by related peptides produced an order of potency of CCK8-sulphated (IC50=5.4 nM)>CCK8-unsulfated (IC50=40 nM) and >CCK4 (IC50=125 nM). The regional distribution of [³H]CCK8S binding in the mouse brain was highest in the olfactory bulb (34.3±5.6 fmol/mg protein) > cerebral cortex > cerebellum > olfactory tubercle > striatum > pons-medulla > mid brain > hippocampus > hypothalamus (12.4±2.1 fmol/mg protein). The repeated administration of haloperidol (2.5 mg/kg/tid) increased the binding of [³H]CCK8S in cerebral cortex from 31.8±1.7 to 38.9±5.2 fmol/mg protein. The varied distribution of CCK8S receptors may signify nonuniform functions for the octapeptide in the brain.     

Modeling of the interaction of Na+ and K+ with the binding of dopamine and [3H]WIN 35,428 to the human dopamine transporter

Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.     

Decreased [3H]ouabain binding sites in skeletal muscle of rats with chronic heart failure

Pickar, Joel G., John P. Mattson, Steve Lloyd, and TimothyI. Musch. Decreased[³H]ouabainbinding sites in skeletal muscle of rats with chronic heart failure.J. Appl. Physiol. 83(1): 323-329, 1997.Abnormalities intrinsic to skeletal muscle are thought tocontribute to decrements in exercise capacity found in individualswith chronic heart failure (CHF).Na⁺-K⁺-adenosinetriphosphatase(the Na⁺ pump) is essential formaintaining muscle excitability and contractility. Therefore, weinvestigated the possibility that the number and affinity ofNa⁺ pumps in locomotor muscles ofrats with CHF are decreased. Myocardial infarction (MI) was induced in8 rats, and a sham operation was performed in 12 rats. The degree ofCHF was assessed ~180 days after surgery. Soleus and plantarismuscles were harvested, and Na⁺pumps were quantified by using a[³H]ouabain bindingassay. At the time of muscle harvest, MI and sham-operated rats weresimilar in age (458 ± 54 vs. 447 ± 34 days old, respectively).Compared with their sham-operated counterparts, MI rats had asignificant amount of heart failure, right ventricular-to-body weightratio was greater (48%), and the presence of pulmonary congestion wassuggested by an elevated lung-to-body weight ratio (29%). Leftventricular end-diastolic pressure was significantly increased in theMI rats (11 ± 1 mmHg) compared with the sham-operated controls (1 ± 1 mmHg). In addition, mean arterial blood pressure was lower inthe MI rats compared with their control counterparts. [³H]ouabain bindingsites were reduced 18% in soleus muscle (136 ± 12 vs. 175 ± 13 pmol/g wet wt, MI vs. sham, respectively) and 22% in plantaris muscle(119 ± 12 vs. 147 ± 8 pmol/g wet wt, MI vs. sham,respectively). The affinity of these[³H]ouabain bindingsites was similar for the two groups. The relationship between thereduction in Na⁺ pump number andthe reduced exercise capacity in individuals with CHF remains to bedetermined.

     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Effects of phospholipids on the specific binding of [3H]spiroperidol to the cholate extract of rat brain synaptic membranes

Effects of phospholipids including PC, PE, PI, and PS on the specific [3H]SPD binding to the solubilized dopamine receptors were examined in the cholate extracts of the cortical and striatal synaptic membranes (P2M) of the rat brain. PC and PS, but not PE or PI, at 0.4 mM greatly enhanced the specific [3H]SPD binding to the cholate extracts of both cortical and striatal P2M fractions. PC and PS did not enhance the specific [3H]DA binding to the same cholate extracts. The enhancing effects of PC and PS were temperature-dependent and in a dose-response manner peaking at 0.4 mM and 0.2 mM respectively. Such temperature dependence indicated that the PC effects were not due to trapping of [3H]SPD by PC but represented a possible DAR-PC complex formation that allowed higher binding for the ligand. Failure of natural cerebellar P2M in enhancing the [3H]SPD binding to the cholate extract supports the notion that fluidity of the phospholipids is required for the binding or the formation of the DAR-PC (or PS) complex. Scatchard analysis of the [3H]SPD binding to the cholate extract in the absence or presence of PC or PS indicated that the PC or PS enhancement of the ligand binding may be mainly due to an increase in the number of binding sites since both PC and PS significantly increased the Bmax but not the Kd of the binding.     

Antihyperglycemic effect of fraxetin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats

Epidemiological studies have demonstrated that the diabetes mellitus is a serious health burden for both governments and healthcare providers. The present study was hypothesized to evaluate the antihyperglycemic potential of fraxetin by determining the activities of key enzymes of carbohydrate metabolism in streptozotocin (STZ) – induced diabetic rats. Diabetes was induced in male albino Wistar rats by intraperitoneal administration of STZ (40 mg/kg b.w). Fraxetin was administered to diabetic rats intra gastrically at 20, 40, 80 mg/kg b.w for 30 days. The dose 80 mg/kg b.w, significantly reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) and increased plasma insulin level. The altered activities of the key enzymes of carbohydrate metabolism such as glucokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and hepatic enzymes (aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP)) in the liver tissues of diabetic rats were significantly reverted to near normal levels by the administration of fraxetin. Further, fraxetin administration to diabetic rats improved body weight and hepatic glycogen content demonstrated its antihyperglycemic potential. The present findings suggest that fraxetin may be useful in the treatment of diabetes even though clinical studies to evaluate this possibility may be warranted.     

Dopamine-stimulated cyclic AMP and binding of [3H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10^?8 M and was half-maximal at $\text{7.9±3.4·10}{}^{\mathrm{?7}}\text{M}$. The increase at 1·10^?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10^?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10^?5 M dopamine was $\text{2.3±0.9·10}{}^{\mathrm{?6}}\text{M}$. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value for high affinity binding sites was $\text{0.43±0.1·10}{}^{\mathrm{?7}}\text{M}$ and $\text{4.7±1.6·10}{}^{\mathrm{?7}}\text{M}$ for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was $\text{1.2±0.4·10}{}^{\mathrm{/t-7}}\text{M}$ noradrenaline, 2·10^/t-7 M epinine, $\text{4.1±1.8·10}{}^{\mathrm{/t-6}}\text{M}$ fluphenazine, $\text{8.0±1.6·10}{}^{\mathrm{/t-6}}\text{M}$ haloperidol, $\text{4.2±1.2·10}{}^{\mathrm{?6}}\text{M}$cis-flupenthixol, $\text{2.7±0.4·10}{}^{\mathrm{?5}}\text{M}$trans-flupenthixol, $\text{>1·10}{}^{\mathrm{?5}}\text{M}$ apomorphine, sulpiride, naloxone and isoproterenol.     

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [³H]-ouabain binding (to analyze K⁺ site of Na⁺, K⁺-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [³H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2 mg/kg) and clozapine (3, 10 and 30 mg/kg). Animals were sacrificed 18 h later, cerebral cortices harvested, membrane fractions prepared and high affinity [³H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10 mg/kg clozapine. Rats were administered with neurotensin (3, 10 y 30 μg, i.c.v.) and 60 min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [³H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30 μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [³H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2 mg/kg, i.p.) or clozapine (10 mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na⁺, K⁺-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na⁺, K⁺-ATPase at central synapses.     

Characterization of [3H]Vesamicol binding in rat brain preparations

The binding of (1)-[³H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P₂ fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P₂ fractions from the cerebral cortex (K_d:21 nM and 980 nM), striatum (K_d:28 nM and 690 nM), and cerebellum (K_d:22 nM and 833 nM). High affinity B_max values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity B_max values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC₅₀:17 nM) and efficaciously (over 90%). Both high affinity and low affinity B_max values for [³H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P₂ fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [³H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [³H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.     

Reduction in [3H]-corticosterone binding to cytoplasmic receptors in the brain of diabetic rats

The binding of [1, 2, 6, 7-³H]-corticosterone was studied in brain cytosol from normal and streptozotocin-diabetic male rats. The experiments were performed under conditions of incubation time (4h), temperature (0–4°C), time after adrenalectomy (6 days) and corticosterone concentrations (1.2 × 10⁻⁸ and 1.15 × 10⁻⁹M) previously established for determining binding activity in the brain of normal rats. The binding of [³H]-corticosterone was found invariably lower in cytosol of the brain from diabetic rats, studied under three different conditions: in non-adrenalectomized animals, in adrenalectomized using a non-saturating corticosterone concentration, and in adrenalectomized plus a saturating steroid concentration. These results support previous contentions that the diminished sensitivity to the negative feedback for steroids which is present in diabetics, may be related to a reduction in binding capacity for corticoids in the central nervous system     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Norharman inhibition of [3H]diazepam binding in mouse brain

Norharman competitively inhibits specific binding of [³H]-diazepam in mouse brain homogenates. $\text{In vivo}$ this β-carboline produces a striking rigid catatonic-like appearance which is abolished by diazepam. It also causes a rapid tremor but has little anticonvulsant effect. Measurement of $\text{in vivo}$ concentrations and receptor occupancy demonstrate that these biological effects occur at doses which occupy a large proportion of benzo-diazepine receptors. It may represent a ligand of the benzo-diazepine receptors whose effects are opposite those of diazepam.     

Compensatory hypertrophy of skeletal muscle fibers in streptozotocin-diabetic rats

Summary Previous studies have demonstrated an apparent differential response of the fiber types in mixed skeletal muscles of rats to streptozotocin diabetes. The purpose of the present study was to examine the ability of the different fiber types to hypertrophy in muscles from diabetic rats, which should further clarify the apparent differential trophic influence of insulin on the fibers. One group of rats was injected with streptozotocin to induce diabetes. The gastrocnemius muscle was then removed from one hindlimb of rats of both the diabetic and a second, normal group, resulting in compensatory growth of ipsilateral plantaris muscle. Rats were sacrificed 60 days following the surgery. Experimental muscles in normal and diabetic rats enlarged 79% and 61% over control muscles, respectively. In normal hypertrophied muscles there was an 8% increase in relative cross-sectional area composed of slow-twitch fibers, whereas in diabetic rats the slow-twitch component increased 17%. The results indicate that slow-twitch fibers in diabetic rats were capable of responding to the chronic power overloaded condition, but that the fast-twitch fibers had a reduced capacity to undergo compensatory growth. These findings support our previous observations suggesting that insulin may exert a differential trophic effect upon the muscle fiber types.Streptozotocin was kindly donated by Dr. W.E. Dulin of the Upjohn Company. This investigation was supported by a Boston University Research Fund Grant     

Specific binding of [3H]phenytoin in the human brain

Competition between cold phenytoin and [3H]phenytoin binding was observed in normal human brain. Binding was observed in all areas examined. The highest number of sites was in the amygdala (a total of 717.71 fmol/mg protein) and the lowest in the Brodman area (BA) 4 of the motor cortex (153.91 fmol/mg protein) and cerebellar cortex (154.4 fmol/mg protein). In three areas, amygdala, cortex area BA 38 (inferior parietal lobe), and cortex area BA 8 (premotor cortex), two sets of binding sites were observed. In these areas the Kd for the higher affinity sites ranged from 35 to 116 nM, and for the lower affinity site, from 328 to 866 nM. In the four areas where only one binding site was observed the KdS ranged from 164 to 311 nM and the Scatchard plot was linear.     

3H]U69,593 binding in guinea-pig brain: comparison with [3H]ethylketazocine binding at the kappa-opioid sites

[3H]U69,593 and [3H]ethylketazocine (mu + delta suppressed) binding was measured in homogenates of guinea-pig brain. Both ligands bind with high affinity to a single class of opioid sites. The relative equilibrium dissociation constant (KD) for [3H]U69,593 is 1.15 nM, while [3H]ethylketazocine has a KD value of 0.33 nM. Their respective maximum binding capacities are 4.49 and 4.48 pmol/g of wet tissue. Various mu-selective, delta-selective, kappa-selective, and nonselective opioids were tested in competition studies against the binding of [3H]U69,593 or [3H]ethylketazocine (in the presence of mu- and delta-blockers) to measure their relative affinity. [D-Ala2, MePhe4,Gly5-ol]enkephalin (mu-selective) has low affinity (600-3000 nM) and [D-Pen2,D-Pen5]enkephalin and [D-Ser2, Leu5, Thr6]enkephalin (delta-selective) have very low affinities (greater than 20,000 nM) at the sites labelled with [3H]U69,593 or [3H]ethylketazocine. On the other hand, unlabelled U69,593, U50,488H, and tifluadom (all three kappa-selective substances) display high affinity (1-5 nM) at those sites. Nonselective opioids, such as bremazocine, levorphanol, and ethylketazocine show similar affinities at the sites labelled with [3H]U69,593 and at the sites labelled with [3H]ethylketazocine. These data indicate that [3H]U69,593 is a selective high-affinity ligand for the same sites that are labelled with [3H]ethylketazocine (in the presence of mu- and delta-blockers) and that these are kappa-sites.