Specific [3H]neurotensin binding to rat spinal cord membranes.

The binding of [3H]neurotensin to membranes prepared from rat spinal cord has been studied in vitro. Scatchard analysis of saturation binding data indicated that [3H]neurotensin binds with high affinity (Kd = 6.3 nM) to a single, saturable population of binding sites (Bmax = 12.4 pmol/g tissue). Neurotensin1-13 (IC50 = 5.9 nM) and neurotensin8-13 (IC50 = 3.7 nM) were potent inhibitors of [3H]neurotensin binding whereas neurotensin1-8 was virtually inactive at concentrations up to 10(-5) M. Sodium chloride (150 mM) significantly inhibited binding, while potassium chloride (5 mM), magnesium chloride (10 mM), manganese chloride (1 mM) and GMP-PNP (0.1 mM) were without effect. The characteristics of the binding of [3H]neurotensin obtained in this study are consistent with this ligand binding to a physiologic neurotensin receptor in rat spinal cord membranes.

Characterization of peripheral-type benzodiazepine recognition sites in the rat spinal cord.

The binding of [3H]Ro 5-4864 to membranes prepared from spinal cord of the adult rat has been studied in vitro. At 4 degrees C, the binding of [3H]Ro 5-4864 reached equilibrium by 120 min, and was rapidly reversible (dissociation t0.5 = 21 min). The [3H]Ro 5-4864 bound with a high affinity (Kd approximately equal to 3 nM) to a single, saturable population of binding sites (Bmax = 27 pmol/g tissue wet weight). Activation of receptors for gamma-aminobutyric acid with 10 microM muscimol did not alter these binding parameters. The drugs Ro 5-4864, diazepam and flunitrazepam were potent inhibitors of this binding (Kis of 10(-9)-10(-8) M) whereas clonazepam, CL 218,872 and Ro 15-1788 were weak inhibitors (Kis greater than 10(-5) M). A comparison of the binding of [3H]Ro 5-4864 in spinal cord with that in other areas of the CNS revealed that whereas the binding affinity was similar in all regions, membranes from spinal cord contained a slightly greater number of binding sites than cerebral cortex and cerebellum, and approximately one-third of the number present in the olfactory bulb. The characteristics of the binding of [3H]Ro 5-4864 obtained in this study are consistent with this ligand binding to peripheral-type benzodiazepine recognition sites in membranes from spinal cord.

Binding of buprenorphine to opiate receptors. Regulation by guanyl nucleotides and metal ions.

The effects of guanosine-5'-triphosphate (GTP), sodium chloride and manganese chloride on the binding of buprenorphine to opiate receptors present in rat brain has been studied. Manganese chloride significantly decreased the affinity of binding of both [3H] buprenorphine and unlabelled buprenorphine to morphine and enkephalin receptors. Guanosine-5'-triphosphate increased the affinity of buprenorphine for morphine sites, but had no effect on binding of buprenorphine to enkephalin or benzomorphan sites, or binding of [3H]buprenorphine. Sodium chloride had no effect on binding of buprenorphine. Control studies indicated similar apparent affinities of buprenorphine for morphine (Ki = 0.30 nM) and enkephalin (Ki = 0.31 nM) sites, and lower affinity for benzomorphan sites (Ki = 4.16 nM). No evidence could be obtained for a differential effect of ions or guanosine-5'-triphosphate on binding of buprenorphine to opiate receptor subtypes.

Characteristics of [3H]fentanyl binding to the opiate receptor.

The present study characterises the binding of the highly lipophilic opiate agonist [3H]fentanyl to homogenates of the rat central nervous system. At 25 degrees C, association of [3H]fentanyl with its binding site was rapid (t1/2 = 2.5 min). Dissociation from the binding site was biphasic (t1/2's = 4.0 and 100 min) suggesting the existence of high and low affinity binding sites. Scatchard plots of saturation isotherms were curvilinear, confirming the presence of high (KD = 0.46 nM) and low KD = 4.26 nM) affinity binding sites. Increasing temperature and the concentration of sodium ion decreased the [3H]fentanyl binding. Opiate agonists, antagonists and mixed agonist-antagonists were all potent (IC50's less than 20 nM) in displacing [3H]fentanyl and displacement by levorphanol and dextrorphan indicated that [3H]fentanyl binding was stereospecific. The mu and delta selective peptides, morphiceptin and [D-Ala2,D-Leu5]enkephalin, had IC50 values of 87 and 9.2 nM respectively. The regional distribution of [3H]fentanyl binding was in the rank order striatum approximately equal to midbrain greater than hypothalamus greater than cortex greater than hippocampus greater than brainstem greater than spinal cord greater than cerebellum. Comparison of [3H]fentanyl, [3H]naloxone and [3H-D-Ala2,D-Leu5]enkephalin binding in the hypothalamus-thalamus (mu-enriched) compared with the frontal cortex-striatum (delta-enriched) indicated that the pattern of [3H]fentanyl labelling was similar to that obtained with [3H]naloxone, but differed from that obtained with [3H-D-Ala2,D-Leu5]enkephalin. These characteristics suggest that [3H]fentanyl binds to the mu-opiate receptor. These findings are discussed in relation to the high lipid solubility of fentanyl as compared with morphine.

Benzodiazepine receptors in the human spinal cord: a detailed anatomical and pharmacological study.

The anatomical distribution and pharmacological characteristics of benzodiazepine receptors in the human spinal cord were examined in four cases aged 20-41 years using in vitro autoradiography and biochemical assays of [3H]flunitrazepam binding. In all cases, the autoradiograms demonstrated that benzodiazepine receptors were distributed in a consistently similar fashion in the gray matter of the cervical, thoracic, lumbar and sacral regions of the human spinal cord. At all levels, the highest densities of benzodiazepine receptors were found to be localized within lamina II of the dorsal horn as defined on cytoarchitectonic, myeloarchitectonic and substance P immunocytochemical criteria. Within this lamina the receptors were concentrated mainly in its deeper, inner portion which lies immediately adjacent to lamina III, with some overlap dorsally into the outer segment of lamina II and ventrally into the adjacent region of lamina III. The lowest density of receptors was found in regions of laminae I, IV, VII and X; in particular, in lamina VII the lowest concentration of receptors was found in the dorsal nucleus of Clarke and the sacral parasympathetic nucleus. The remaining laminae of the spinal gray (laminae, V, VI, VIII and IX) showed a moderate density of receptors. Biochemical assays of membranes prepared from the lumbosacral cord indicated that these [3H]flunitrazepam binding sites have high affinity and have the pharmacological characteristics of the "central" Type II benzodiazepine receptor. These results show a high concentration of Type II benzodiazepine receptors in the substantia gelatinosa of the human spinal cord and suggest a possible role for these receptors in spinal sensory functions.

Opiate receptors in the human spinal cord: a detailed anatomical study comparing the autoradiographic localization of [3H]diprenorphine binding sites with the laminar pattern of substance P, myelin and nissl staining.

The anatomical localization of opiate receptors in the human spinal cord has been examined in six cases aged 7-41 years using quantitative autoradiographic methods following the incubation of fresh, unfixed cryostat sections with [3H]diprenorphine. In order to precisely localize the distribution of receptors in the spinal cord, the laminar anatomy of the spinal grey was demonstrated at each spinal level examined using 50-microns sections stained for myelin, Nissl substance and substance P. In all cases, autoradiograms demonstrated that opiate receptors were distributed in a similar fashion in the grey matter of the cervical, thoracic, lumbar, sacral and coccygeal regions of the human spinal cord. At all 25 spinal levels examined, opiate receptors were mainly localized within the upper laminae of the dorsal horn (laminae I-III) and within the tract of Lissauer. The highest density of opiate receptors was localized within the inner segment of lamina II where the receptors formed a very dense band lying immediately dorsal to lamina III. The density of receptors in this inner region of lamina II (33 +/- 2 fmol/mg) was more than two-and-one-half times greater than that in the remaining upper laminae which showed moderate receptor densities: lamina I (12 +/- 4 fmol/mg) and outer lamina II (13 +/- 3 fmol/mg) both showed similar receptor densities which were higher than those in lamina III (10 +/- 3 fmol/mg) The tract of Lissauer (11 +/- 2 fmol/mg) also showed a moderate density of opiate receptors which was intermediate between the densities in laminae I/IIo and the density of lamina III. The density of receptors in the remaining laminae of the spinal cord varied from moderately low to virtually zero. Moderately low densities of receptors were found in laminae V, VI, VIII, IX and X with very low levels within laminae IV and VII. In particular, in lamina VII opiate receptors were unable to be detected above normal background levels in the dorsal nucleus of Clarke. These results show that, as in other mammalian species, opiate receptors in the human spinal cord are mainly concentrated in the upper laminae of the dorsal horn and in the tract of Lissauer. The possible role of these receptors in modulating spinal nociceptive information is discussed with respect to previous findings on the relationship of opiate receptors to primary afferent fibres in the spinal cord.

Benzodiazepine receptors in the human hippocampal formation: a pharmacological and quantitative autoradiographic study.

The pharmacological characteristics and anatomical distribution of benzodiazepine receptors in the human hippocampal formation were studied in seven cases aged 4-68 years. The pharmacology of the receptors was studied by computerized, non-linear least squares regression analysis of [3H]flunitrazepam displacement by flunitrazepam, CL218,872 and ethyl beta-carboline-3-carboxylate binding to membranes and the anatomical localization of these receptors was demonstrated using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam. The pharmacological studies indicated that the human hippocampal formation contained equal numbers of benzodiazepine receptors with high affinity (Type I) and low affinity (Type II) for CL218,872 and ethyl beta-carboline-3-carboxylate. The autoradiograms demonstrated that the benzodiazepine receptors were distributed in a heterogeneous fashion throughout the major regions of the human hippocampal formation; the highest concentrations of receptors were present in the dentate gyrus (molecular layer) and field CA1 of Ammon's horn (strata pyramidale, oriens, lacunosum), with moderate concentrations in field CA2 of Ammon's horn (stratum pyramidale) and in regions of the subicular complex and entorhinal cortex, and with considerably lower densities in fields CA3 and CA4. Quantitative analyses of the autoradiograms showed that the regions containing the highest densities of receptors (molecular layer of dentate gyrus and the strata oriens, pyramidale and lacunosum of CA1) were enriched with Type 1 receptors whereas other regions of lower receptor densities were enriched with either Type I or Type II receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple benzodiazepine receptors in the human basal ganglia: a detailed pharmacological and anatomical study.

The pharmacological characteristics and anatomical distribution of benzodiazepine receptors in the striatum (dorsal striatum, comprising the caudate nucleus and putamen, and ventral striatum) and globus pallidus (dorsal pallidum, comprising the external and internal segments, and ventral pallidum) of the human basal ganglia were examined in twelve cases aged 4-71 years. The pharmacology of the receptors was studied using computerized, non-linear least-squares regression analysis of [3H]flunitrazepam displacement by flunitrazepam, CL218,872 and ethyl beta-carboline-3-carboxylate binding to membranes. The results showed that the dorsal striatum (caudate nucleus and putamen) contained higher concentrations of receptors than the dorsal pallidum (external and internal segments). The dorsal striatum contained equal numbers of sites with high affinity (Type I) and low affinity (Type II) for CL218,872 and ethyl beta-carboline-3-carboxylate whereas the globus pallidus contained sites with only high affinity (Type I) for these ligands. The anatomical localization of the benzodiazepine receptor subtypes (Type I and II) was studied using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam in the absence or presence of the discriminating ligand CL218,872. The autoradiograms showed that benzodiazepine receptors were distributed throughout all regions of the human striatum in a heterogeneous fashion, i.e. high-density patches of receptors were set against a background matrix of lower receptor densities. The highest densities of receptors were seen in the ventral striatum where the patches were particularly extensive and showed densities 56% higher than the receptor densities in the dorsal striatal patches. Quantitative analysis showed that the patches in all striatal regions contained mainly Type II receptors (83%-86%) whereas the matrix regions in the ventral and dorsal striatum contained different proportions of the receptor subtypes; Type I receptors predominated (60%) in the matrix of the ventral striatum and Type II receptors predominated (67%-71%) in the matrix of the dorsal striatum. By contrast, the autoradiograms showed that the globus pallidus contained considerably lower concentrations of receptors than the striatum. The highest density of receptors in the globus pallidus was present in the ventral pallidum with successively lower concentrations in the external (26% less) and internal (66% less) segments of the dorsal pallidum. In agreement with the membrane binding studies the receptors in the globus pallidus were mainly of the Type I variety.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotensin receptors in the human spinal cord: a quantitative autoradiographic study.

The anatomical localization of neurotensin receptors in the human spinal cord was examined in 12 cases aged 4-68 years using quantitative autoradiographic methods following the incubation of fresh, unfixed cryostat sections with 4 nM [3H]neurotensin. Characterization of the pharmacological specificity of the [3H]neurotensin binding sites in the human spinal cord from displacement studies with neurotensin and various neurotensin fragments indicated that, whereas 1.0 microM neurotensin and the carboxy-terminal fragment neurotensin almost completely displaced [3H]neurotensin binding (4 nM), the amino-terminal fragments neurotensin and neurotensin1-11 were weak inhibitors. This requirement for the carboxy-terminal fragment neurotensin is consistent with [3H]neurotensin binding to specific neurotensin receptors in the human spinal cord. In all cases the autoradiograms demonstrated that neurotensin receptors were distributed in a similar fashion in the gray matter of the cervical, thoracic, lumbar, sacral and coccygeal regions of the human spinal cord. At all 21 spinal levels examined, the highest density of neurotensin receptors was localized in lamina II of the dorsal horn. Within lamina II the receptors were especially concentrated in the deeper inner segment (IIi) where they formed a dense band lying immediately dorsal to lamina III. The density of receptors in this inner region of lamina II (23.5 fmol/mg) was almost double that in the outer segment of lamina II (12.2 fmol/mg), which showed the next highest density of receptors, and more than three times that in the adjacent lamina I (6.9 fmol/mg) and lamina III (7.1 fmol/mg). A moderate density of receptors was present in the intermediomedial (8.0 fmol/mg) and intermediolateral (8.0 fmol/mg) nuclei of lamina VII, and in lamina IX (4.4 fmol/mg). The density of labelling in the remaining laminae of the spinal cord was very low. These results indicate that neurotensin receptors are mainly localized in somatic and visceral sensory and motor regions of the human spinal cord and suggest that neurotensin may play a role in modulating sensory-motor functions in the human spinal cord.

Testing cyclic AMP mediation of memory: reversal of alpha-methyl-p-tyrosine-induced amnesia.

Treatments that increased intracellular cyclic 3',5' adenosine monophosphate (cAMP) levels following catecholamine depletion caused by alpha-methyl-p-tyrosine (AMPT) provided a prophylactic effect against AMPT-induced amnesia. This effect gives evidence that cAMP mediated the formation of memory. In Experiment I, the phosphodiesterase inhibitor papaverine (50 mg/kg), immediately after a one-trial acquisition task, functionally increased cAMP levels and prevented amnesia 3 h after treatment with AMPT (200 mg/kg) for New Zealand A strain (NZ/A) mice tested in a step-through passive avoidance apparatus. Retention test latencies 72 h later were significantly higher for animals that received only saline and for animals that received AMPT and papaverine than for animals that received AMPT and saline (the amnesic group). In a similar task (Experiment II), mice that received an intracerebroventricular injection of either 5 or 10 microgram dibutyryl cAMP immediately after acquisition and 3 h after AMPT administration showed significantly higher retention test latencies than animals that received AMPT and saline. The AMP plus 10 microgram dibutyryl cAMP group showed facilitated performance even compared to the saline plus saline group.

Heterogeneous distribution of benzodiazepine receptors in the human striatum: a quantitative autoradiographic study comparing the pattern of receptor labelling with the distribution of acetylcholinesterase staining.

The distribution of benzodiazepine receptors in the human striatum was studied by quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam, and the pattern of receptor-labelling was compared to the distribution of acetylcholinesterase (AChE) staining in adjacent sections. A heterogeneous pattern of benzodiazepine receptors was found in all regions of the striatum. The highest densities of receptors were seen in the ventral striatum (nucleus accumbens and olfactory tubercle), where very dense receptor patches aligned with both AChE-poor and AChE-rich regions. The dorsal striatum (caudate nucleus and putamen) contained lower concentrations of benzodiazepine receptors, but dense receptor patches were still evident (especially in the caudate nucleus) and these aligned with AChE-poor striosomes.

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding sites.

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [3H]quinuclidinylbenzilate (QNB) and [3H]pirenzepine (PZ) followed by autoradiography. The highest densities of [3H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [3H]PZ binding was localized to lamina II where the binding density was 22-32% higher than in lamina IX. These results suggest that the M1 and M2 muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.

The distribution of neurotensin receptors and acetylcholinesterase in the human caudate nucleus: evidence for the existence of a third neurochemical compartment.

The distribution of neurotensin receptors in the human caudate nucleus was studied using autoradiographic methods following in vitro labelling of cryostat sections with [3H]neurotensin, and the pattern of receptor labelling was compared to the distribution of acetylcholinesterase (AChE) staining in adjacent sections. A heterogeneous pattern of neurotensin receptors was found in the caudate nucleus. Patches of low receptor density aligned with the AChE-poor striosomes, regions of moderate receptor density corresponded with the AChE-rich matrix zone, and annular regions of high receptor density aligned with the AChE-negative border zone lying between the AChE-poor striosome and the AChE-rich matrix compartments. These results suggest the existence of 3 neurochemical compartments within the human caudate nucleus.

Benzodiazepine receptors in the human cerebellar cortex: a quantitative autoradiographic and pharmacological study demonstrating the predominance of type I receptors.

The anatomical localization of benzodiazepine receptors in the human cerebellar cortex was studied using quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam ([3H]FNZ), and the pharmacology of these receptors has been characterized by computerized, non-linear least squares regression analysis of [3H]FNZ displacement by FNZ, CL218,872 and ethyl beta-carboline-3-carboxylate (ECC) binding to membranes. The autoradiograms demonstrated that benzodiazepine receptors were present throughout all layers of the human cerebellar cortex; high concentrations of receptors were present in the molecular layer, moderate concentrations were present in the granular layer and a much lower density of receptors was seen in the intervening Purkinje cell layer. The pharmacological studies indicated that the human cerebellar cortex contained a high concentration of homogeneous benzodiazepine receptors which have high affinity for FNZ, ECC and CL218,872, i.e. type I sites.

Specific [3H]Ro 5-4864 binding to rat spinal cord membranes: evidence for peripheral type benzodiazepine recognition sites.

The binding of [3H]Ro 5-4864 and [3H]methylclonazepam to membranes prepared from adult rat spinal cord has been studied in vitro. Scatchard analysis of saturation isotherms suggested that both 3H-labeled ligands bind to single binding sites ([3H]Ro 5-4864 Kd = 2 nM, [3H]methylclonazepam Kd = 3.5 nM), although [3H]Ro 5-4864 bound to 3 times the number of sites labeled by [3H]methylclonazepam (respective Bmax values were 15 vs 5.3 pmol/g tissue). Displacement experiments with clonazepam, flunitrazepam and Ro 5-4864 indicated that [3H]Ro 5-4864 and [3H]methylclonazepam binding had the expected pharmacologic specificity for peripheral and central benzodiazepine recognition sites respectively (i.e. [3H]methylclonazepam binding was sensitive to clonazepam but not Ro 5-4864 whereas [3H]Ro 5-4864 binding was potently inhibited by Ro 5-4864 but not clonazepam. Flunitrazepam had similar affinities for both sites). Thus, in addition to central type benzodiazepine receptors, the rat spinal cord contains comparatively high concentrations of peripheral benzodiazepine recognition sites.

A comparison of the new topical antibiotic mupirocin ('Bactroban') with oral antibiotics in the treatment of skin infections in general practice.

A trial was carried out in general practice in 200 patients presenting with skin infections to compare topical antibiotic treatment with mupirocin ointment with orally administered flucloxacillin or erythromycin. Patients were assigned at random to receive 4 to 10 days' treatment with either mupirocin applied 3-times daily or one of the oral antibiotics in the dosage normally used by the general practitioner for skin infections. The majority of infections were impetigo and infected wounds/lacerations; the main organisms isolated initially from 127 of the patients were either Staphylococcus aureus or beta-haemolytic Group A streptococci. Clinical response to mupirocin ointment (86% cured, 13% improved) was significantly better than that seen with erythromycin (47% cured, 26% improved) and similar to that with flucloxacillin (76% cured, 23% improved). Treatment outcome was not related to treatment duration with either the topical or oral preparations. Post-treatment samples from 76 patients showed that in the mupirocin group all the pathogens originally isolated were eliminated, including Gram-negative organisms.

Morphiceptin: biphasic inhibition of mu-opiate receptor ligands.

The interaction between morphiceptin and the morphine (mu) opiate receptor present in rat brain membranes has been examined. Detailed competitive displacement curves of morphiceptin against the mu receptor ligands [3H]fentanyl and [3H]naloxone were biphasic, with Hill coefficients of 0.78 and 0.60 respectively. Hoftsee plots of these displacement curves suggested that 30-35% of the morphiceptin binding was to a high affinity site and the residual binding was to a site with lower affinity. These results indicate that morphiceptin binding to the mu opiate receptor does not obey the law of mass action, and raises the possibility that morphiceptin distinguishes subclasses of mu binding site.

Characteristics of type 1 and type 2 benzodiazepine receptors in the ovine brain.

Analysis of the displacement of 3H-diazepam binding to membranes prepared from the ovine frontal cortex by the triazolopyradiazine CL218,872 yielded a Hill coefficient significantly below unity. By analogy with similar studies of this drug in rat brain this suggested the existence of Types 1 and 2 benzodiazepine receptors. The degree of displacement of 3H-diazepam by CL218,872 (200 nM, Type 1; 800 nM, Type 2) in homogenates of brain regions differed, the rank order being cerebellum greater than parietal cortex greater than frontal cortex congruent to temporal cortex congruent to hippocampus greater than striatum. Displacement of 3H-diazepam by CL218,872 was enhanced by 10(-5) M GABA in the striatum (at 200 nM and 800 nM CL218,872) and cerebellum (at 200 nM CL218,872). Benzodiazepine receptors in the ovine frontal cortex were least sensitive to CL218,872 (200 nM) in young fetuses (54-68 days gestation) and achieved adult levels of sensitivity by late gestation. Finally, the potency of CL218,872 to displace 3H-diazepam was not effected by the 3H-ligand concentration (0.5 nM or 5.0 nM), suggesting that Types 1 and 2 benzodiazepine receptors are not identical to the high and low affinity 3H-diazepam binding sites we have previously identified in the ovine brain.