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PubMed1987

Cytotoxic T lymphocyte recognition of a xenogeneic major histocompatibility complex antigen expressed in transgenic mice.

Introduction of a porcine major histocompatability complex (MHC) class I gene (PD1) into the genome of a C57BL/10 (B10) mouse has been shown to lead to cell surface expression of the porcine MHC antigen, SLAPD1 in a transgenic mouse. The PD1 product expressed on spleen cells from the transgenic mice stimulated B10 spleen cells in a mixed lymphocyte culture to generate PD1-specific cytotoxic T lymphocytes (CTL). The CTL were PD1 specific since they lysed transgenic splenic blast cells and PD1-transfected L cells, but not B10 blasts or control L cells. The CTL were L3T4-, Lyt-2+ and their activity was partially inhibited by either anti-Lyt-2 antibody or by anti-swine MHC alloantibodies. The repertoire of responding B10 anti-transgenic CTL was assessed by examining their cross-reactivity on a series of murine allogeneic targets. The B10 anti-transgenic CTL showed some cross-reactivity on conventional allogeneic targets, but reacted strongly on a series of mutant H-2Kbm blast cells. In addition, B10 anti-B6.cH-2bm6 CTL cross-reacted extensively on the transgenic target cells. These results demonstrated that normal B10 CTL possess a repertoire specific for the products of the xenogeneic class I gene PD1, that this repertoire is cross-reactive with the conventional alloreactive CTL repertoire, and that there exists an unanticipated relationship between PD1-specific CTL and CTL specific for Kb mutant determinants.

Animals↗

PubMed1989

Expression of a class I MHC transgene: effects of in vivo alpha/beta-interferon treatment.

Transgenic mice containing a swine class I major histocompatibility complex (MHC) gene, PD1, express swine MHC (SLA) antigen. The tissue distribution of PD1 RNA parallels that observed in the swine, indicating that the expression of PD1 is regulated and that trans-acting factors involved in this regulation have been conserved between the species. Although PD1 RNA levels were much greater in transgenic spleen than in thymus, no difference in the chromatin organization of the PD1 gene was detected. In both tissues, a single DNase I hypersensitive site mapped within the 5' flanking region. In vivo treatment of the transgenics with mouse alpha, beta-interferon increases PD1 expression in a number of tissues. In the spleen, this increase parallels that observed for the endogenous transplantation antigen, Kb, but differs markedly from the differentiation antigen, Qa-2. Increases in cell surface expression of both PD1 and Kb occurred equally in splenic T- and B-cell populations following alpha, beta-interferon treatment. In contrast, Qa-2 expression in B cells was enhanced by alpha, beta-interferon, whereas it was unaffected in T cells and thymocytes.

Animals↗

PubMed1990

Postnatal development of the electrical activity of rat nigrostriatal dopaminergic neurons.

Extra- and intracellular recordings were obtained in vivo from dopaminergic nigrostriatal neurons in rat pups ranging in age from postnatal day (PD) 1 to PD28, and in adult rats. Neurons from PD1-3 rats were active at very low rates in a random pattern, rarely showed bursting activity, and often exhibited long periods of up to several minutes of silence. Spontaneous spikes were of relatively low amplitude and long duration. The mean firing rate increased and became more regular over time, and short bursts consisting of only 2 spikes were observed. By the second postnatal week, the initial segment component of the spontaneous spike resembled that seen in adults, but the somadendritic component was still relatively small, and there was often a very marked temporal delay between the two. Near the end of the second postnatal week, neurons exhibited a transient phase of pacemaker-like activity. Mean firing rates continued to increase with time, as did the incidence and complexity of bursting activity. The spontaneous firing rate, pattern and spike morphology approached adult values by the fourth postnatal week. Antidromic responses from neostriatum were obtained as early as PD1, and consisted of a significantly greater proportion of full initial segment-soma dendritic spikes compared to nigrostriatal neurons from adult rats. There was usually a long delay between the initial segment and somadendritic components of the spike. Mean antidromic latency and mean antidromic threshold did not vary significantly from PD1-3 to adults. Axonal conduction velocity reached maximal adult values by PD16-21. Neostriatal-evoked orthodromic responses consisted principally of a poststimulus inhibition whose duration decreased from PD1 through adulthood. Pure excitatory responses were very rarely observed at any age. Intracellular recordings from PD2, PD3 and PD5 rats revealed striatal-evoked inhibitory postsynaptic potentials in non-dopaminergic nigral neurons with a mean onset latency (9.8 +/- 3.8 ms) which did not differ from that previously reported for adult rats.

Action Potentials↗

PubMed1988

Identification of negative and positive regulatory elements associated with a class I major histocompatibility complex gene.

Regulatory DNA sequence elements were functionally identified in the 5'-flanking region of a gene, PD1, which encodes a porcine classical transplantation antigen. Both a positive regulatory element and a novel negative regulatory DNA element were mapped within 1.1 kilobases upstream of exon 1. The negative regulatory element reduced the activity of both the homologous PD1 promoter and a heterologous simian virus 40 promoter. In vivo competition experiments indicated that the functions of the PD1 positive and negative regulatory elements are mediated by distinct cellular trans-acting factors. The PD1 positive regulatory element interacted with cellular factors in common with those binding to the simian virus 40 enhancer. Finally, the negative regulatory element required the presence of a positive regulatory element to function. This interaction between positive and negative regulatory elements represents a novel mechanism for regulating gene expression.

Amino Acid Sequence↗

PubMed1990

Light-dependent D1 protein synthesis and translocation is regulated by reaction center II. Reaction center II serves as an acceptor for the D1 precursor.

Light induces an irreversible modification of the photosystem II reaction center (RCII) affecting specifically one of its major components, the D1 protein (Ohad, I., Adir, N., Koike, H., Kyle, D. J., and Inoue, Y. I. (1990) J. Biol. Chem. 265, 1972-1979) which is degraded and replaced continuously (turnover). The turnover rate of D1 is related to light intensity. Evidence is presented showing that RCII translocates from the site of damage in the grana (appressed) domain of the chloroplast membranes to unappressed membrane domains where the D1 precursor protein (pD1) is translated and becomes integrated into RCII. Several forms of RCII (a, a*, and b) were identified on the basis of their electrophoretic mobility. pD1 was found only in the a and b forms in the unappressed membranes. Processing of pD1 occurs after its integration into RCII. Mature D1 appeared mostly in the a form of RCII and following its translocation to the appressed membrane domains also in the a* form. Thus the light intensity-dependent synthesis of D1 protein is related to the availability of modified RCII which serves as an acceptor for pD1. The shuttling of RCII between the two membrane domains may represent a control mechanism of thylakoid membrane protein synthesis.

Biological Transport↗

PubMed1987

Structure and expression of class I MHC genes in the miniature swine.

The genome of the miniature swine, unlike other species, contains a relatively small class I MHC gene family, consisting of only seven members. This provides an excellent system in which to identify and characterize the regulatory mechanisms which operate to both coordinately and differentially regulate the expression of a multi-gene family. The structure of class I SLA genes, like other class I genes, consists of eight exons encoding a leader sequence, three extracytoplasmic domains, a transmembrane domain and intracytoplasmic domains. Despite the common structure, two sub-families of class I genes can be distinguished within the SLA family. One, containing the closely related PD1 and PD14 genes, encodes the classical transplantation antigens. Another contains the highly divergent PD6; the functions of the products of this subfamily, if any, are not known. The class I SLA genes share some common regulatory mechanisms, as evidenced by the fact that all three genes analyzed are transcribed in mouse L cells. Furthermore, interferon treatment of transfected mouse L cells enhances expression of all three genes. Both PD1 and PD6 are transcribed in vivo, where the highest levels of expression are observed in lymphoid tissues. Superimposed on the common patterns of class I gene expression are distinct ones, as evidenced by the findings that PD1 is preferentially expressed in B cells, whereas PD6 is preferentially expressed in T cells. These differences may reflect the extensive divergence of the 5' flanking sequences of these genes. Future studies will be aimed at elucidating the precise molecular interactions and mechanisms which give rise to the observed differential expression.

Animals↗

PubMed2020

Combination of cyclin-dependent kinase and immune checkpoint inhibitors for the treatment of bladder cancer.

BACKGROUND: Perturbation of the CDK4/6 pathway is frequently observed in advanced bladder cancer. We investigated the potential of targeting this pathway alone or in combination with chemotherapy or immunotherapy as a therapeutic approach for the treatment of bladder cancer METHODS: The genetic alterations of the CDK4/6 pathway in bladder cancer were first analyzed with The Cancer Genome Atlas database and validated in our bladder cancer patient-derived tumor xenografts (PDXs). Bladder cancer cell lines and mice carrying PDXs with the CDK4/6 pathway perturbations were treated with a CDK4/6 inhibitor palbociclib to determine its anticancer activity and the underlying mechanisms. The combination index method was performed to assess palbociclib and gemcitabine drug-drug interactions. Syngeneic mouse bladder cancer model BBN963 was used to assess whether palbociclib could potentiate anti-PD1 immunotherapy. RESULTS: Of the 413 bladder cancer specimens, 79.2% harbored pertubations along the CDK4/6 pathway. Palbociclib induced G0/G1 cell cycle arrest but with minimal apoptosis in vitro. In mice carrying PDXs, palbociclib treatment reduced tumor growth and prolonged survival from 14 to 32 days compared to vehicle only controls (p = 0.0001). Palbociclib treatment was associated with a decrease in Rb phosphorylation in both cell lines and PDXs. Palbociclib and gemcitabine exhibited antagonistic cytotoxicity in vitro (CI > 3) and in vivo, but palbociclib significantly enhanced the treatment efficacy of anti-PD1 immunotherapy and induced CD8+ T lymphocyte infiltration in syngeneic mouse models. CONCLUSIONS: The CDK4/6 pathway is feasible as a potential target for the treatment of bladder cancer, especially in combination with immunotherapy. A CDK4/6 inhibitor should not be combined with gemcitabine.

Animals↗

PubMed1992

The origins of supraspinal projections to the cervical and lumbar spinal cord at different stages of development in the gray short-tailed Brazilian opossum, Monodelphis domestica.

We have used the retrograde transport of Fast blue (FB) to study the origins of supraspinal projections to the lumbar and cervical spinal cord at different stages of development in the Brazilian, short-tailed opossum, Monodelphis domestica. Monodelphis was chosen for study because its young are born in a very immature state, 14-15 days after copulation, making it possible to manipulate its nervous system in an embryonic state without intra-uterine surgery. When injections of FB were made into the lumbar cord at postnatal day (PD) 1, neurons were labeled within several areas of the reticular formation (the retroambiguus nucleus, the ventral and dorsal reticular nuclei of the medulla, the gigantocellular reticular nucleus, the lateral paragigantocellular reticular nucleus, and the pontine reticular nucleus), the presumptive coeruleus complex, and the lateral vestibular nucleus. In many cases, labeled neurons were also found within the caudal raphe and the presumptive interstitial nucleus of the medial longitudinal fasciculus. The results of immunocytochemical studies provided evidence for catecholaminergic and serotoninergic neurons in the brainstem at PD1 and for axons of both phenotypes in the spinal cord. By PD3, labeled neurons were found within the ventral gigantocellular and ventral pontine nuclei of the reticular formation, the spinal trigeminal nucleus, and the presumptive paraventricular nucleus of the hypothalamus. When injections were made at PD4, neurons were also labeled within the medial and inferior vestibular nuclei, the red nucleus, the mesencephalic nucleus of the trigeminal nerve, the presumptive nucleus of Edinger-Westphal and the lateral hypothalamus. By at least PD7, the pattern of supraspinal labeling was similar to that obtained at older ages and in the adult animal. When FB was injected into the cervical cord at PD1, neurons were labeled in all of the areas labeled by lumbar injections at the same age and in larger numbers. In addition, labeled neurons were found within the ventral gigantocellular and spinal trigeminal nuclei. When cervical injections were made at PD15, labeled neurons were found within the deep cerebellar nuclei and amygdala and by PD17 they were also present within the superior colliculus and cerebral cortex. In some cases, cortical labeling was present outside the areas labeled by comparable injections in adult animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Amidines↗

PubMed1991

Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.

Blotting, Southern↗

PubMed1990

Expression of a class I MHC transgene: regulation by a tissue-specific negative regulatory DNA sequence element.

In vivo patterns of expression of a miniature swine class I major histocompatibility gene, PD7, were analyzed both in situ in the pig, and in transgenic mice. Structural analysis of PD7 DNA sequences revealed that PD7 is highly homologous to the pig gene PD1, which encodes a classical transplantation antigen. Despite the extensive homology, PD7 is expressed in situ at markedly lower levels than PD1 in nearly all tissues. Introduction of PD7 into mice results in a pattern of PD7 expression in the transgenic animals that parallels that observed in situ in the pig. Comparison of two lines of PD7 transgenic mice, which differ only in the extent of 5' flanking sequence, reveals the presence of a silencer element. The silencer activity is tissue specific: differences in PD7 expression are observed only in lymphoid tissues and skin. Skin from both lines of transgenics mediates graft rejection, but the rate of rejection correlates with the level of PD7 expression.

Amino Acid Sequence↗

PubMed1992

Long-term survival of mouse corpus callosum grafts in neonatal rat recipients, and the effect of host sensitization.

Previous studies have suggested that the incidence of spontaneous rejection among immunogenetically mismatched neural transplants in neonatal recipients varies significantly depending on the cellular composition of the graft material. For example, neuron-rich grafts of embryonic mouse retina generally survive for extended periods without showing signs of rejection after implantation into neonatal rats, whereas cortical xenografts, which contain abundant glial and endothelial cells as well as neurons, typically undergo rejection 4-6 weeks after implantation. To determine whether the presence of donor glia is responsible for this high incidence of spontaneous rejection, we examined the fate of a non-neuronal graft material composed predominantly of xenogeneic glial cells (post-natal day 3, PD3, CD-1 mouse corpus callosum) implanted into the mesencephalon of PD1 Sprague-Dawley rats. The distribution and survival of donor astrocytes were assessed using a monoclonal antibody specific for a mouse astrocyte surface antigen, M2. Thirteen of 16 animals sacrificed within 2 months of implantation had detectable transplants. In these animals, M2-positive cells frequently migrated well away from body of the graft, clustering in large numbers in several characteristic regions of the host brain. Unlike cortical grafts of similar age, the vast majority (93%) of callosal transplants showed no histological signs of rejection or major histocompatibility complex antigen expression in and around the transplant-derived cells. As previously noted in the neonatal retinal transplant paradigm, however, well-integrated 1-month-old corpus callosum grafts could be induced to reject by appropriate sensitization of the host immune system, implying that the host was not immunologically tolerant to the foreign neural graft. With longer survival times in unsensitized hosts, a progressively smaller percentage of animals had detectable donor astrocytes (5 of 10 animals at 3 months postimplantation and 4 of 16 animals at 4 months); in those 9 animals with surviving grafts, only small numbers of M2-positive cells were seen within the graft bed and surrounding host brain. However, only 2 of the 26 "long-term" animals showed evidence of graft rejection. These results indicate that mouse astrocytes show characteristic patterns of migration into the host brain when implanted into neonatal rats; however, these xenogeneic cells have a limited duration of survival. The infrequency with which even subtle signs of spontaneous rejection were detected in animals that had received corpus callosum xenografts suggests that an immune-mediated process is unlikely to be responsible for the time-dependent elimination of the donor astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl- transport.

The rat cortical collecting duct (CCD) exhibits high rates of NaCl reabsorption when stimulated by mineralocorticoid and antidiuretic hormone (ADH). The present study was undertaken to determine if there is significant transcellular Cl- movement across the principal cells of the rat CCD. CCDs were dissected from kidneys of rats that had been injected with deoxycorticosterone (5 mg, i.m.) 2-9 days prior to the experiment. The ducts were perfused in vitro with identical perfusing and bathing solutions, except that 200 pmol.l-1 ADH was added to the bathing solutions. The basolateral membrane voltage (PDbl) of principal cells was -77 +/- 1 mV and the luminal membrane voltage (PD1) was -68 +/- 1 mV (mean +/- SEM, n = 124). Separate impalements with single-barrelled Cl(-)-selective microelectrodes gave an apparent intracellular Cl- activity of principal cells of 17 +/- 2 mmol.l-1. Transepithelial PD and PDbl were unaffected by luminal furosemide, hydrochlorothiazide (HCT), 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid, (SITS), or the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); bath addition of SITS or the Cl- channel blocker diphenylamino-2-carboxylic acid; or replacement of bath HCO3- by Cl-. The intracellular Cl- activity (a(cell)Cl) also remained unchanged with the addition of HCT, SITS or the Cl- channel blockers to either the perfusing or bathing solutions, or with replacement of the bathing solution HCO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1991

The complete amino acid sequence of prolactin from the bullfrog, Rana catesbeiana.

The complete amino acid sequence of prolactin (PRL) from an amphibian species, the bullfrog (Rana catesbeiana), has been determined and conserved residues and domains were analyzed by sequence comparison of PRLs from 15 species of five major vertebrate classes. The bullfrog PRL consists of 197 amino acid residues with three disulfide linkages formed between residues 4-11, 58-172, and 189-197. The bullfrog PRL shows the highest identity with sea turtle PRL (75%); lower identities with chicken PRL (72%), pig, horse, and fin whale PRLs (68%), human, cattle, sheep, and elephant PRLs (60-58%), and rat and mouse PRLs (50%); and significantly lower identity with teleost PRLs (about 30%). It is apparent that all tetrapod PRLs characterized so far contain three disulfide bonds in homologous positions and differ from teleost PRLs which lack the N-terminal disulfide loop. The tetrapod and teleost PRLs share 34 common residues and these conserved residues are clustered in six domains (PD1 to PD6), suggesting that these common residues, or at least part of them, are responsible for the activities common to all PRLs. On the other hand, PD5 is conserved significantly within tetrapod PRLs, but to a lesser extent in teleost PRLs, suggesting that the PD5 contributes to the activities specific to tetrapod PRLs.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of prenatal cocaine on behavioral responses to a cocaine challenge on postnatal day 11.

The purpose of this study was to investigate the effects of prenatal cocaine treatment on behavioral responsivity to a cocaine challenge on postnatal day (PD) 11. Timed-pregnant Sprague-Dawley rats were injected s.c. with 40 mg/kg/day of cocaine (20 mg/kg twice daily) from gestational day 11 to 20. Saline-control females received saline injections and were pair-fed to the cocaine-treated females, whereas untreated-control females were undisturbed and were fed ad lib. Litters were culled to eight pups on PD1 and fostered to normal lactating dams. On PD 11, subjects were given either saline or cocaine (1.25, 2.5, or 5.0 mg/kg s.c.) and then tested 15 min later for isolation-induced ultrasonic vocalizations and other behaviors. Prenatal cocaine-treated pups showed a reduced sensitivity to the stimulating effect of postnatal cocaine on wall climbing, which may reflect an underlying alteration in central dopaminergic and/or noradrenergic systems. Most of the other behaviors studied, including ultrasonic vocalizations, were unaffected by prenatal cocaine administration. However, one other notable finding was an increase in postnatal mortality among the cocaine-exposed pups. We hypothesize that prenatal cocaine treatment may alter pup behavior so as to produce abnormal maternal-offspring interactions and impaired development in some individuals.

Animals↗

PubMed2025

Population analysis and immunologic landscape of melanoma in people living with HIV.

PURPOSE: To dissect the clinical and immunological features of people living with HIV (PLWH) diagnosed with melanoma, who have consistently exhibited worse clinical outcomes than HIV-negative individuals (PLw/oH) with the same cancer. EXPERIMENTAL DESIGN: We analyzed electronic health records from 1,019 PLWH and 373,121 PLw/oH diagnosed with melanoma. Demographic and clinical characteristics were compared. Spatial immune transcriptomics (72 immune-related genes) was performed on melanoma tumor samples (n=11), followed by downstream validation using multiplex immunofluorescence (n=15 PLWH, n=14 PLw/oH). RESULTS: PLWH were diagnosed with melanoma at a younger age, had a higher representation of Hispanic and Black individuals compared to PLw/oH, and a decreased survival rate. PLWH also showed a markedly increased risk of brain metastases. PLWH experienced significant delays in initiating immune checkpoint inhibitor (ICI) therapy and had worse survival outcomes following ICI, even after balancing for demographic covariates. Spatial transcriptomics revealed a more immunosuppressive tumor microenvironment in PLWH, with upregulation of immune checkpoints (PD1, LAG3) and reduced expression of antigen presentation markers (HLA-DRB, B2M), with distinct spatial distributions in tumors and their microenvironments. Multiplex immunofluorescence confirmed an exhausted CD8+ T cell compartment in PLWH, including enrichment of PD1intLAG3- and PD1intLAG3+ subpopulations, and a significant accumulation of immunosuppressive myeloid-derived suppressor cells (CD11b+ HLA-DR- CD33+). CONCLUSIONS: Our findings suggest chronic HIV infection fosters a permissive tumor microenvironment that might undermine effective immune responses and contribute to poor clinical outcomes for PLWH with melanoma. Targeting the actionable immune pathways identified in this study could inform tailored therapeutic strategies to mitigate these disparities.

HIV↗

PubMed1988

PD117302: a selective agonist for the kappa-opioid receptor.

1. A new nonpeptide kappa-opioid compound, a cyclohexyl benzeneacetamide derivative (PD117302), has been synthesized and its affinity for the different types of opioid receptor determined. The ability of PD117302 to modify the activity of the electrically-stimulated guinea-pig ileum and rabbit vas deferens has also been evaluated. 2. In binding studies using guinea-pig brain homogenates, unlabelled PD117302 had a high affinity (Ki = 3.7 nM) at [3H]-etorphine labelled kappa sites and a low affinity at [3H]-[D-Ala2, MePhe4, glyol5]-enkephalin ([3H]-DAGOL) labelled mu sites (Ki = 408 nM) and [3H]-SKF 10047 labelled sigma sites (Ki = 1.8 microM). In bioassay studies, PD117302 was a potent agonist, producing a maximum inhibition of the electrically-evoked contractions of the guinea-pig ileum (IC50 = 1.1 nM) and rabbit vas deferens (IC50 = 45 nM) which was naloxone-reversible. 3. In guinea-pig brain, [3H]-PD117302 bound to a high-affinity opioid binding site with a KD of 2.7 nM and a Bmax of 3.4 pmol g-1 wet weight. The Bmax was found to be less than 50% of the Bmax values for [3H]-etorphine and [3H]-bremazocine suggesting that [3H]-PD117302 may be a specific ligand for a subtype of kappa receptor. [3H]-PD117302 also bound with micromolar affinity to a non-opioid binding site. 4. Kinetic studies found that [3H]-PD117302-specific binding to the high affinity site was saturable, reaching equilibrium within 20 min at 4 degrees C, and reversible, with a half-life of dissociation of 3.9 min. 5. Several unlabelled compounds with high affinities for the ['H]-etorphine labelled K binding site, had comparable affinities when competing for the ['H]-PDl 17302-specific high affinity binding site. In contrast, DAGOL, [D-Ala', D-Leu'] enkephalin (DADLE) and [D-Pen2, D-Pen'] enkephalin (DPDPE) had no significant effect on ['H]-PD 1 17302 binding, suggesting minimal interaction with mu and delta binding sites. 6. In autoradiography studies ['H]-PD1 17302 binding sites were found throughout the brain with the greatest density in the striatum, cerebral cortex (layers V-VI), substantia nigra, and the molecular layer of the cerebellum. Lowest levels were found in the granular layer of the cerebellum, thalamus and cerebral cortex (layers I - IV).

Animals↗

PubMed1990

Opioid receptor ligands in the neonatal rat spinal cord: binding and in vitro depression of the nociceptive responses.

1. Opioid receptors in the neonatal rat spinal cord have been characterized by measurements of ligand binding to crude membrane fractions and by functional tests on the nociceptive spinal response in a spinal cord-tail preparation in vitro. 2. There were high affinity binding sites for [3H]-[D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGOL), [3H]-U69593, and [3H]-ethylketocyclazocine (EKC) on spinal cord membranes from neonatal rats. Hill slopes for binding of [3H]-DAGOL and [3H]-U69593 were close to unity. The Hill slope for binding of [3H]-EKC was less than unity, even after its interactions at mu-receptors had been blocked with 100 nM unlabelled DAGOL. Binding sites for [3H]-[D-Pen2, D-Pen5]enkephalin (DPDPE) could not be detected. 3. In competition assays U50488 was as potent as PD117302 and U69593 in competition for either [3H]-U69593 or [3H]-EKC binding sites. Hill slopes for a range of competing ligands at [3H]-DAGOL or [3H]-U69593 sites were close to unity. Hill slopes for competition at [3H]-EKC sites were less than one. 4. In the spinal cord-tail preparation from neonatal rats, opioid receptor agonists depressed spinal nociceptive responses evoked by application of capsaicin or heat to the tail. The order of potency was DAGOL greater than U69593 = PD117302 greater than morphine greater than U50488 = [D-Pen2, L-Pen5]enkephalin (DPLPE). 5. The antagonist naloxone was about equally potent against DAGOL, morphine and DPLPE, and about ten times less potent against U69593 and PD117302. The effects of U50488 were much less sensitive to blockade by naloxone than the effects of PD11703 or U69593. The Kappa antagonist, nor-binaltorphimine was equipotent against all three Kappa agonists. 6. The absence of delta-binding sites, and the low potency and relatively high sensitivity to naloxone suggest that DPLPE could be working at mu-receptors in the neonatal rat spinal cord. 7. The binding assays show that U50488 has the same affinity as PD1 17302 and U69593 for Kappa-receptors, yet it was less effective in the depression of nociceptive responses. This may be because U50488 has a relatively low efficacy at Kappa-receptors. It is possible that at high concentrations U50488 activates receptors not affected by other Kappa-ligands. These additional receptors may be non-opioid receptors (hence the insensitivity to naloxone), or they could be a subtype of Kappa-opioid receptor.

Animals↗

PubMed1991

Pharmacological profile of various kappa-agonists at kappa-, mu- and delta-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the rat brain.

1. The potency, relative efficacy and selectivity of a series of kappa-opioid receptor agonists at the mu-, delta- and kappa-opioid receptors mediating inhibition of electrically-induced (radiolabelled) neurotransmitter release from superfused rat brain slices was determined. 2. With regard to their potencies at kappa-receptors mediating inhibition of striatal [3H]-dopamine release, the highest pD2 value (8.7) was found for bremazocine and the lowest (7.1) for U50488; the pD2 values for ethylketocyclazocine (EKC), tifluadom, U69593 and PD117302 were between 8.0 and 8.3. There were no marked differences between the relative efficacies of the kappa-agonists (maximum inhibition being 60-70%). In contrast to the other kappa-agonists, at a concentration of 1 microM, PD117302 caused a significant (25-40%) increase of the spontaneous efflux of tritium. 3. None of the kappa-agonists significantly affected striatal [14C]-acetylcholine (ACh) release, with the exception of a slight inhibitory effect of EKC. The delta-receptor-mediated inhibitory effect of [D-Ala2, D-Leu5]enkephalin (DADLE) on [14C]-ACh release was antagonized in a concentration-dependent manner by bremazocine (0.1 and 1.0 microM) and also partially by EKC (1 microM), but not by the other kappa-agonists. The pA2 value for bremazocine as an antagonist at the delta-receptors involved was 8.0, compared to 7.6 for naloxone. 4. None of the kappa-agonists significantly affected cortical [3H]-noradrenaline (NA) release, with the notable exception of tifluadom, which strongly inhibited release by activating mu-receptors. The mu-receptor-mediated inhibitory effect of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) on [3H]-NA release was antagonized in a concentration-dependent manner by bremazocine and EKC, but not by the other K-agonists. The pA2 value for bremazocine as an antagonist at the mu-receptors involved was 8.2, compared to 8.6 for naloxone. 5. Thus, whereas U69593 and PD1 17302 display high potency and selectivity towards K-opioid receptors, the potent benzomorphan K-agonists bremazocine and EKC also appear to be strong mu-opioid receptor antagonists.

Acetylcholine↗