Opiate radioreceptor assay distinguishes between anesthetics and convulsants: the effect of pH

The specific binding of [³H]naloxone to brain tissue is perturbed by anesthetics and convulsants in a rank order of potency related to lipophilicity of the drugs. Neurodepressants can be distingished from neuroexcitants in the assay, because sodium ion enhances the action of the former and antagonizes the action of the latter. As the pH of the assay medium is varied from 6.7 to 7.7 to 8.4, perturbation of [³H]naloxone binding by the neurodepressants, diethyl ether and Isoindoklon, is unaffected either in the presence or absence of sodium. In contrast, the antagonism by sodium of the neuroexcitants, DDT and Indoklon (the convulsant isomer of Isoindoklon), is progressively enhanced with increasing pH. These observations on the effects of pH extend earlier ones on ion effects and should contribute to the delineation of specific membrane loci that mediate the actions of neurodepressants as distinct from sites that mediate the actions of neuroexcitants.     

Opiate receptor binding in the brain of the hypertensive rat

Experimental and genetic hypertension in male rate is accompanied by a lower specific [3H]naloxone binding in the dorsal horn of the spinal cord, and in the hippocampus as compared to controls. Rats which are genetically resistant to hypertensive stimuli have a higher specific [3H]naloxone binding in the nucleus tractus solitarius and lower opiate receptor binding in the dorsal horn. Together with previous studies which demonstrated a correlation between blood pressure and pain sensitivity, these results support the notion that specific brain loci participate in co-regulation of pain perception and blood pressure.     

Opiate antagonist receptor binding in vivo: evidence for a new receptor binding model

The in vivo accumulation and retention of the opiate antagonist tracers [3H]diprenorphine and [3H]naloxone at cerebral opiate receptor sites in rats exceed that expected from their known in vitro receptor affinities. The [3H]diprenorphine serum and brain levels can be stimulated with a pharmacokinetic model that contains the receptors in a micro-compartment. The receptor micro-compartment consists of a population of binding sites next to a diffusion boundary which restricts ligand diffusion away from the receptor. Such an arrangement introduces a delay in the binding equilibrium of potent antagonists with the receptor sites and an increase in the apparent in vivo receptor affinity at subsaturating doses of the ligand; at saturating ligand concentrations these functions of the receptor micro-compartment are abolished. A physiological interpretation of the receptor micro-compartment could be the location of clustered opiate receptor sites on the exterior cell surface next to the synaptic cleft as the diffusion boundary. This kinetic approach involving a combination of pharmacokinetics and drug-receptor interactions permits the quantitative analysis of receptor site availability in the intact animal. Our results support the hypothesis that only one receptor population affects the in vivo disposition of the antagonist tracers, while they do not exclude the presence of low affinity binding sites that have been observed with the use of [3H]naloxone in vitro. Moreover, the binding site population observed in vivo may be responsible for mediating opiate agonist analgesia.     

Opiate receptor binding-effect relationship: Sufentanil and etorphine produce analgesia at the μ-site with low fractional receptor occupancy

The analgesic activity of the opiate agonists etrophine and sufentanil and the antagonistic effects of diprenorphine and naloxone have been related to the occupancy of 3 classes of opiate binding sites previously defined in vivo²⁹in order to establish their pharmacological significance. Sufentanil binds specifically in vivo to the first type of site (site 1), exhibiting 1100-fold selectivity over site 2, whereas etorphine displays 20-fold selectivity for site 1 over site 2. Neither agonist has measurable affinity to the third type of binding site. The binding data suggest that site 1 is analogous to the μ site previously identified in vitro²⁹. Both agonists produce analgesia in the rat tail flick test at the same low fractional occupancy of site 1 ( 2% at the ED₅₀) while they display much lower and quite different occupancies at site 2. Both of the opiate antagonists naloxone and diprenorphine reduce the potency of sufentanil and etorphine by a factor of 2 at 50% occupancy of site 1 alone. These results provide strong evidence that these 4 drugs exert their effects by interaction with site 1 (μ sites) which therefore may be regarded as the receptor responsible for analgesic action in this test. The assumption of a direct relationship between antagonistic effect and fractional occupancy appears to be valid for naloxone and diprenorphine at site 1, while the agonists exert their action at a very low fractional occupancy implying a non-linear binding-effect process.     

Opiate and opioid peptide binding in rat goldfish: further evidence for opiate receptor heterogeneity

The binding of a series of 3H-labeled mu, kappa, sigma and delta opioid agonists and an antagonist has been examined in rat and goldfish brain membranes. A variety of treatments, including NaCl, N-ethylmaleimide, trypsin and chymotrypsin show dramatic differences both between the two species and between the different 3H-ligands.     

[H]-etorphine and [H]-diprenorphine receptor binding in vitro and in vivo: differential effect of Na and guanylyl imidodiphosphate

The in vivo and in vitro cerebral receptor binding kinetics of the opiate agonist etorphine and the antagonist diprenorphine were investigated in the rat. Although of similar receptor affinity in vitro in Tris buffer brain homogenates, etorphine exhibited considerably less affinity than diprenorphine in vivo. The hypothesis was tested whether the opiate receptor regulators, Na⁺ and GTP, are responsible for the low in vivo receptor affinity of the agonist. [³H]Etorphine and [³H]diprenorphine dissociation curves were similarly affected by Na⁺ and guanylyl imidodiphosphate (GPP(NH)P), a hydrolysis resistant GTP analog when added separately in vitro. However, the combination of Na⁺ and GPP(NH)P greatly accelerated only the [³H]etorphine off-rate over that with Na⁺ alone and reproduced the rapid dissociation half-life observed in vivo (t_1/2 < 1 min). In contrast, the receptor dissociation rate of the antagonist was not further accelerated by Na⁺ plus GPP(NH)P over that with Na⁺ alone. Moreover, Na⁺ plus GPP(NH)P decreased [³H]etorphine, but not [³H]diprenorphine, equilibrium binding in vitro. These results suggested that the lower in vivo affinity of etorphine than of diprenorphine is predominantly caused by the combined action of Na⁺ and GTP. Furthermore, the data are consistent with the hypothesis that both etorphine and diprenorphine bind to the opiate receptor in its high affinity form, but that only the agonist etorphine is capable of converting the high affinity form to one of low affinity in the presence of Na⁺ and guanine nucleotide. Assuming that production of the low affinity form reflects biological activation of the opiate receptor system, then this hypothesis is consistent with the high pharmacological potency of etorphine (agonistic ED₅₀ 1μg/kg) relative to its low apparent in vivo receptor affinity, as well as with the low fractional receptor occupancy of etorphine ( 2%) at its analgesic ED₅₀. Finally, in vivo [³H]etorphine and [³H]diprenorphine displacement curves obtained with unlabeled diprenorphine and etorphine showed that [³H]etorphine labels only a subpopulation of the total [³H]diprenorphine binding sites. It remains to be determined which subsites of the opiate receptor system mediate the agonistic actions of etorphine.     

3H]-etorphine and [3H]-diprenorphine receptor binding in vitro and in vivo: differential effect of Na+ and guanylyl imidodiphosphate

The in vivo and in vitro cerebral receptor binding kinetics of the opiate agonist etorphine and the antagonist diprenorphine were investigated in the rat. Although of similar receptor affinity in vitro in Tris buffer brain homogenates, etorphine exhibited considerably less affinity than diprenorphine in vivo. The hypothesis was tested whether the opiate receptor regulators, Na⁺ and GTP, are responsible for the low in vivo receptor affinity of the agonist. [³H]Etorphine and [³H]diprenorphine dissociation curves were similarly affected by Na⁺ and guanylyl imidodiphosphate (GPP(NH)P), a hydrolysis resistant GTP analog when added separately in vitro. However, the combination of Na⁺ and GPP(NH)P greatly accelerated only the [³H]etorphine off-rate over that with Na⁺ alone and reproduced the rapid dissociation half-life observed in vivo (t_1/2 < 1 min). In contrast, the receptor dissociation rate of the antagonist was not further accelerated by Na⁺ plus GPP(NH)P over that with Na⁺ alone. Moreover, Na⁺ plus GPP(NH)P decreased [³H]etorphine, but not [³H]diprenorphine, equilibrium binding in vitro. These results suggested that the lower in vivo affinity of etorphine than of diprenorphine is predominantly caused by the combined action of Na⁺ and GTP. Furthermore, the data are consistent with the hypothesis that both etorphine and diprenorphine bind to the opiate receptor in its high affinity form, but that only the agonist etorphine is capable of converting the high affinity form to one of low affinity in the presence of Na⁺ and guanine nucleotide. Assuming that production of the low affinity form reflects biological activation of the opiate receptor system, then this hypothesis is consistent with the high pharmacological potency of etorphine (agonistic ED₅₀ ～ 1μg/kg) relative to its low apparent in vivo receptor affinity, as well as with the low fractional receptor occupancy of etorphine (～ 2%) at its analgesic ED₅₀. Finally, in vivo [³H]etorphine and [³H]diprenorphine displacement curves obtained with unlabeled diprenorphine and etorphine showed that [³H]etorphine labels only a subpopulation of the total [³H]diprenorphine binding sites. It remains to be determined which subsites of the opiate receptor system mediate the agonistic actions of etorphine.     

Monoclonal antibodies that distinguish between normal and denervated human acetylcholine re receptor

Ten monoclonal anti-human acetylcholine receptor (AChR) monoclonal antibodies (m.abs) all exhibited high avidity binding to the human AChR. None was able to inhibit α-bungarotoxin (α-Butx) binding to the receptor.Five distinct but partially overlapping antibody-binding regions were defined by competition experiments. Four antibodies, which competed with each other for one region on denervated human AChR and also bound to human fetal AChR, failed to bind appreciably to normal human AChR in solution, to normal AChR solubilized from 6 other species, or to human endplates in frozen sections.     

Evidence for additional neurotensin receptor subtypes: neurotensin analogs that distinguish between neurotensin-mediated hypothermia and antinociception

Neurotensin (NT), a tridecapeptide, is a neurotransmitter that elicits potent effects including hypothermia and antinociception in mice and rats. To date, there are two types of the neurotensin receptor (NTR) that have been molecularly cloned from the rat. However, several lines of evidence suggest the presence of additional NTR subtypes. We have identified a NT analog of the NT(8-13) fragment, NT27, that selectively causes only the hypothermic response in vivo, when microinjected into the periaqueductal gray (PAG) of rats. A dose of 18 nmol of NT or NT27 caused a body temperature lowering of 1.8 and 1.2°C, respectively. This same dose of NT or NT27 yielded a hotplate maximum physiological effect of 75% and 25%, respectively. Interestingly, despite its high K_D (620 nM) at the cloned NTR-1, NT27-I (the iodinated form of NT27) exerted a potent hypothermic effect even at a very low dose (0.6 nmol). Equally intriguing, was that NT24, a sterioisomer of NT27, with a much higher affinity (K_D=0.5 nM) at NTR-1, did not selectively induce hypothermia in mice, but did selectively induce hypothermia in rats.     

The Possible Role of Protein Phosphatase 2A in the Sodium Sensitivity of the Receptor Binding of Opiate Antagonists Naloxone and Naltrindole

In striatal membrane preparation used for receptor binding experiments high levels of protein phosphatase 1 and 2A activities were detected using [³²P]phosphorylase a as substrate. Sodium chloride decreased the activity of protein phosphatase 2A and increased the activity of protein phosphatase 1 in a concentration-dependent manner. Sodium chloride facilitated the saturation binding of naloxone and naltrindole in rat striatal membrane preparation preincubated with ATP (50 μM) and MgCl₂ (5 mM). Preincubation with calyculin A (1 nM) further increased the binding of naloxone. Addition of okadaic acid in a concentration of 2 nM, which is specific for the inhibition of protein phosphatase 2A, augmented the number of binding sites of naloxone or naltrindole. The results suggest a protein phosphatase-dependent regulation of the binding of opiate ligands in the striatum.     

Regulation of opiate receptors in mouse brain: arcuate nuclear lesion induces receptor up-regulation and supersensitivity to opiates

Lesion of the hypothalamic arcuate nucleus of the mouse by neonatal application of monosodium glutamate (MSG) increased the binding of [³H]dihydromorphine to membranes prepared from the midbrain. A saturation curve of [³H]dihydromorphine binding indicated that MSG increased the number of the opiate receptors. The MSG-treated mice also exhibited an enhanced response to morphine and naltrexone regarding thermal pain sensitivity. The physiological implications of opiate receptors up-regulations upon arcuate nuclear lesion are discussed.     

Difference in response of D2 receptor binding between11C-N-methylspiperone and11C-raclopride against anesthetics in rhesus monkey brain

Summary The effects of anesthesia on dopamine D₂ receptor binding in the rhesus monkey brain were examined using positron emission tomography. The bindings of¹¹C-N-methylspiperone (NMSP) and¹¹C-raclopride (RAC) were measured under controlled ketamine or isoflurane anesthesia. The binding of¹¹C-NMSP was significantly lower in the striatum anesthetized with isoflurane than with ketamine. There was a smaller change in the binding of¹¹C-RAC than of¹¹C-NMSP. These findings suggest that changes in¹¹C-NMSP or¹¹C-RAC binding induced by anesthetics were not due solely to changes in the competition of endogenous dopamine.     

III. Comparison of the antinociceptive action of Mu and delta opioid receptor ligands in the periaqueductal gray matter, medial and paramedial ventral medulla in the rat as studied by the microinjection technique

In rats stereotaxically implanted with microinjection cannula in either the periaqueductal gray matter (PAG) or the medial/paramedial medullary reticular formation (MRF), microinjection of morphine, sufentanil,d-Ala²-d-Leu⁵-enkephalin (DADL) ord-Ser²- Thr⁶-leucine enkephalin (DSTLE) produced dose-dependent elevations in the response latency on tail-flick and hot plate tests. These effects were reversed by naloxone administered by microinjection into the same intracerebral site. Both mu (morphine and sufentanil) and delta (DADL and DSTLE) opioid receptor ligands produced a maximal elevation in the supraspinally mediated hot plate response when administered into either the PAG or the MRF. Similarly, mu and delta receptor ligands produced maximum elevations in the spinally mediated tail-flick response when microinjected into the PAG. In contrast, delta, but not mu, receptor agonists produced a total blockade of the tail-flick response following administraion into the MRF. Microinjection of mu (morphine) or delta (DADL) agonists into the PAG or the MRF also resulted in a naloxone-reversible inhibition of the visceral chemical evoked writhing response. These observations suggest that mu and delta opioid receptor linked systems within the MRF but not the PAG produce their antinociceptive effects by discriminable mechanisms with a differential action on spinopetal vs supraspinal modulatory systems.     

Colocalization and shared distribution of endomorphins with substance P, calcitonin gene-related peptide, gamma-aminobutyric acid, and the mu opioid receptor

The endomorphins are endogenous opioids with high affinity and selectivity for the mu opioid receptor (MOR, MOR-1, MOP). Endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2); EM1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2); EM2) have been localized to many regions of the central nervous system (CNS), including those that regulate antinociception, autonomic function, and reward. Colocalization or shared distribution (overlap) of two neurotransmitters, or a transmitter and its cognate receptor, may imply an interaction of these elements in the regulation of functions mediated in that region. For example, previous evidence of colocalization of EM2 with substance P (SP), calcitonin gene-related peptide (CGRP), and MOR in primary afferent neurons suggested an interaction of these peptides in pain modulation. We therefore investigated the colocalization of EM1 and EM2 with SP, CGRP, and MOR in other areas of the CNS. EM2 was colocalized with SP and CGRP in the nucleus of the solitary tract (NTS) and with SP, CGRP and MOR in the parabrachial nucleus. Several areas in which EM1 and EM2 showed extensive shared distributions, but no detectable colocalization with other signaling molecules, are also described.     

Association of a polymorphism of the serotonin 1B receptor gene and alcohol dependence with inactive aldehyde dehydrogenase-2

Summary. The use of persons who become alcoholic despite having a well-defined negative risk for alcoholism (inactive aldehyde dehydrogenase-2 or ALDH2) is advantageous in genetic research because of this population's reduced heterogeneity and possible genetic factors conferring susceptibility to alcohol dependence. This investigation of central serotonin neurotransmission, specifically the serotonin 1B (5HT1B) receptor gene and its role in both regulating alcohol consumption and developing alcohol dependence revealed overrepresentation of the C allele of the 861G>C polymorphism of 5HT1B in alcoholics with inactive ALDH2, compared with its frequency in nonalcoholic controls. No significant differences in 5HT1B genotype and allele distributions were observed between alcoholics with active ALDH2 and controls, however. Taken together with recent observations, these results suggest that genetic variability of the 5HT1B receptor is involved in the development of some type of alcohol dependence. Received October 10, 2001; accepted November 9, 2001     

D2-dopamine receptor occupancy differs between patients with and without extrapyramidal side effects

To investigate whether the occurrence of extrapyramidal side effects was related to D2 dopamine receptor occupancy, iodobenzamide single positron emission computed tomography was carried out in 27 schizophrenic patients and 10 controls. Eighteen patients were treated with haloperidol; 9 patients were treated with clozapine. Our data suggest a relationship between D2 receptor occupancy and extrapyramidal side effects as well as the existence of a neuroleptic threshold of a striatal:frontal cortex ratio of 1.2, below which drug-induced exptrapyramidal side effects can be expected.