Human growth factor for murine interleukin (IL)-9 responsive T cell lines: co-induction with IL-6 in fibroblasts and identification as LIF/HILDA.

Two mouse helper T cell clones that proliferate in response to murine interleukin (IL)-9 could also be grown in conditioned medium of stimulated human connective tissue cells. The activity was not due to known T cell growth factors including human IL-9, which is not effective on mouse cells. This growth-stimulatory activity for TS1 cells (GATS) was co-induced with IL-6 on normal fibroblasts and certain sarcoma cell lines stimulated with IL-1, double-stranded RNA, virus or phorbol ester. However, the conditions for optimal induction and the kinetics of production were found to be different for IL-6 and GATS. GATS from phorbol ester-stimulated human hepatosarcoma cells co-purified with IL-6, but could be separated from it by subsequent cation-exchange fast-protein liquid chromatography and reverse-phase high-performance liquid chromatography. Homogeneous tumor cell-derived GATS was a 25-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas IL-6 produced by these cells appeared in its 23-kDa form. Pure GATS was found to be inactive in the B cell hybridoma growth assay for IL-6. Finally, GATS was identified by NH2-terminal sequence analysis of the mature protein as leukemia inhibitory factor or human interleukin for DA cells (LIF/HILDA). The effect of LIF/HILDA on T cells was not mediated by IL-2, IL-4 or IL-9 production. Since this cytokine has not previously been reported to act on T cells, further investigation of its role in T cell activation should be taken into consideration.


High amounts of circulating interleukin (IL)-6 in the form of monomeric immune complexes during anti-IL-6 therapy. Towards a new methodology for measuring overall cytokine production in human in vivo.

A patient with plasma cell leukemia was treated with anti-interleukin (IL)-6 monoclonal antibodies (mAb) for 2 months. Using chromatography on protein A-Sepharose, anti-murine-IgG-Sepharose, anti-IL-6-mAb-Sepharose and gel filtration at pH 2.3, we have demonstrated that the anti-IL-6 mAb, by preventing the binding of IL-6 to its cell membrane receptor and its renal elimination, has induced huge amounts of IL-6 to circulate in the form of monomeric immune complexes. By using this observation, we have developed a mathematical modelling that allows the determination of the overall daily production of IL-6 in this patient, which was in the range of 15 micrograms per day. Overall in vivo production of cytokines has never been evaluated in animals or in humans before.


Classical transplantation tolerance in the adult: the interaction between myeloablation and immunosuppression.

Allogeneic bone marrow transplantation in the neonate is an effective way of inducing permanent tolerance to donor tissue. To do the same in the immunocompetent adult requires immunosuppression to counter host-versus-graft alloreactivity. Conditioning with monoclonal antibodies (mAb) to CD4 and CD8 has been sufficient where donor and recipient are mismatched at only multiple "minor" histocompatibility loci, or at major histocompatibility complex (MHC) class I plus "minor" loci, but not where the mismatch involves the entire MHC. Tolerance across the MHC barrier requires extra conditioning with agents that happen to be both immunosuppressive and myeloablative, so obscuring the assessment of which effect is important. By using dimethylmyleran as a selective "space"-creating myeloablative agent, and CD4 plus CD8 mAb as sole immunosuppressive agents, we have been able to dissect the relative requirements for immunosuppression and myeloablation. We show here that transplantation tolerance could only be achieved when both types of agent were combined together so as to guarantee sufficient donor-type hemopoietic chimerism. We argue that the donor marrow, given sufficient space, will engraft and provide a sustained source of tolerogen overriding any host resistance that antibodies cannot control.


Expression and biological activity of interleukin-1 receptors in mouse gamma/delta thymocytes during ontogeny.

We have recently shown that the response of mouse thymocytes to interleukin (IL)-1 + IL-2 was maximal at birth and that the responding cells displayed a CD4-CD8- T cell receptor (TcR) gamma/delta + phenotype. Unexpectedly, despite their high proportion of gamma/delta + cells, fetal thymocyte populations responded only weakly to IL-1 + IL-2. In this report, we demonstrate that the discrepancy between the day 17.5 fetal and newborn sensitivities to the combined action of IL-1 and IL-2 is a consequence of the different patterns of high-affinity IL-1 receptor (IL-1R) expression displayed by these two cell subsets. Actually, high- and low-affinity IL-1R are found in TcR gamma/delta + newborn cells and, in contrast, only low-affinity IL-1R are detectable in day 17.5 fetal cells. Our binding and functional studies strongly support the hypothesis that high-affinity IL-1R on the one hand, and low-affinity ones on the other hand, are involved in the response to IL-1 + IL-2 of newborn and day 17.5 fetal thymocytes, respectively. In addition, the high-affinity IL-1R appear to be far more efficient than the low-affinity receptors in promoting IL-2 responsiveness of thymocytes.


In vivo administration of interleukin-2 turns on anergic self-reactive T cells and leads to autoimmune disease.

One major mechanism of self tolerance involves the deletion of T cell clones in the thymus. In athymic mice, tolerance to self antigens must be generated extrathymically. T cells with self-reactive receptors undergo either peripheral clonal deletion or become unresponsive (i.e. anergic). The unresponsive state of human and mouse T cell clones in vitro can be reversed by the addition of exogenous interleukin (IL)-2, thus transforming anergic T cells to an activated state. Here it is shown that the in vivo delivery of IL-2 to athymic BALB/c nu/nu mice abrogates the anergic state of self-reactive V beta 3+ and V beta 11+ T cells [which are normally deleted in the minor lymphocyte stimulatory (Mls)-1b-, I-E(+)-expressing euthymic counterparts]. Thus, V beta 3+ and V beta 11+ T cells from IL-2-treated nude mice proliferate in response to T cell receptor cross-linking and acquire effector functions as measured by their ability to deliver aid to B cells upon specific stimulation. This activation correlates with the development of autoimmune manifestations (DNA autoantibodies, rheumatoid factors, erythroleukopenia and minimal change nephritis) in these IL-2-treated mice.


Interferon-gamma induced lethality in the late phase of Plasmodium vinckei malaria despite effective parasite clearance by chloroquine.

A combination therapy was tested consisting of chloroquine and interferon-gamma (IFN-gamma) in the late phase of blood-stage Plasmodium vinckei malaria in BALB/c mice. When mice were treated with three times 300 micrograms chloroquine at 24-h intervals starting at a parasitemia of 30%-50%, only 5 of 14 mice (36%) died 2-4 days after initiation of therapy. However, when infected mice received chloroquine plus 1 microgram IFN-gamma at the same time, 14 of 18 mice (78%) died 0.5-3 days after start of therapy (p < 0.05) despite clearance of parasitemia. The histopathology from mice dying after combination therapy revealed interstitial leukocyte infiltration of lung tissue, severe liver cell necrosis and kidney tubular necrosis. Pretreatment of P. vinckei-infected mice with pentoxifylline, a phosphodiesterase inhibitor, led to a significant decrease of IFN-gamma-induced lethality (p < 0.05). In contrast, pretreatment with neutralizing antibodies to tumor necrosis factor or with L-N-monomethyl arginine, the latter an inhibitor of the nitric oxide synthase, significantly increased lethality (p < 0.05).


Molecular analysis of anti-idiotypic monoclonal antibodies in the HLA-DR antigenic system.

The structural organization of anti-idiotypic (id) antibodies has been investigated mostly in haptenic systems. No information is available about the structural characteristics of anti-id antibodies in major histocompatibility complex (MHC) antigenic systems, although these data may contribute to our understanding of the molecular basis of their functional role in the immune response. Therefore, we have determined the nucleotide and derived amino acid sequence of the VH and VL regions of the anti-id monoclonal antibodies (mAb) F5-444, F5-830, F5-963, F5-1126, F5-1336 and F5-1419, which had been elicited with the syngeneic anti-HLA-DR1, 4, w14, w8, 9 mAb AC1.59. The six anti-id mAb are heterogenous in their VH and VL region gene usage. This structural heterogeneity is not correlated with their target specificity and with their ability to elicit anti-HLA-DR antibodies. The latter characteristic is markedly influenced by a limited number of amino acid substitutions, since mAb F5-444, which induces anti-HLA-DR antibodies, differs only in two residues in complementarity-determining regions and in five residues in framework regions from mAb F5-1126, which does not induce anti-HLA-DR antibodies. The heterogeneity in VH and VL region gene usage by the six anti-id mAb in the HLA-DR system is at variance with the restricted VH and VL region gene usage by syngeneic anti-id mAb in several haptenic systems. Furthermore, at variance with haptenic systems, the primary structure of the D segments of the anti-id mAb is not correlated with their ability to induce anti-HLA-DR antibodies. On the other hand, the frequency of D-D fusion events underlying the derivation of the D segments of the six anti-id mAb in the HLA-DR system and their average length are similar to those found in anti-id mAb in haptenic systems. In addition, like in the latter systems, somatic mutations appear to contribute to the generation of diversity of anti-id mAb in the HLA-DR system.


Molecular characterization of the V gamma 9 T cell receptor repertoire expressed in patients with rheumatoid arthritis.

We have characterized the variable (V)gamma 9 T cell receptor (TcR) repertoire expressed in synovial membrane (SMC) and peripheral blood mononuclear cells (PBMC) of five rheumatoid arthritis (RA) patients. The experimental approach was to sequence the junctional regions of polymerase chain reaction (PCR)-amplified V gamma 9 transcripts; gamma chain sequences were compared with those found in normal PBMC. For normal PBMC, a large proportion of V gamma 9 transcripts used the joining (J)gamma P gene segment whereas a few used J gamma 2. Despite this restriction in J gamma usage, there was extensive junctional region diversity with a unique sequence observed in each transcript examined. In contrast to normal PBMC, J gamma usage of V gamma 9 transcripts in PBMC of two patients was skewed towards J gamma 2. This deviation in J gamma usage was more pronounced in SMC since all patients expressed V gamma 9 transcripts in SMC which predominantly used J gamma 2, as opposed to J gamma P in normal PBMC. Further, approximately 60% of V gamma 9 transcripts in PBMC of each of three patients had identical junctional region sequences, although the specific sequences were unique in each patient. For two of these patients the dominant transcript found in the PBMC was detected in the corresponding SMC at about 10% and 40%, respectively. Overall, our findings indicate that V gamma 9-bearing T cells in RA peripheral blood are largely derived from clonal expansion whereas in the synovium there is expression of a population of these cells that are mainly polyclonal. This may reflect in vivo expansion in response to a V gamma 9/J gamma 2 region-specific antigen. The data presented in this report suggest that V gamma 9+ T cells may play a role in the development of RA.


Signal transduction for interleukin-3-dependent leukotriene synthesis in normal human basophils: opposing role of tyrosine kinase and protein kinase.

The intracellular signaling pathways regulating the synthesis of leukotrienes by myeloid cells are largely unknown. In addition, the signal transduction mechanisms utilized by the cytokine receptor family are still poorly understood. The fact that in mature human basophils the synthesis of leukotriene C4 (LTC4) induced by C5a is strictly dependent on a short preincubation with the cytokine interleukin-3 (IL-3), allowed us to investigate the metabolic requirements for LTC4 synthesis, and also to provide some information on early signal transduction mechanisms of IL-3 in these differentiated, non-dividing blood leukocytes. IL-3 itself does not alter intracellular free calcium concentration ([Ca2+]i) in basophils, whereas C5a induces a transient rise independent of IL-3 pretreatment, indicating that the priming effect of IL-3 cannot be explained by alterations in [Ca2+]i changes. The protein kinase C inhibitor staurosporine did not inhibit C5a-induced histamine release nor IL-3-dependent LTC4 formation in contrast to the IgE receptor-dependent basophil response. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) induced histamine release without leukotriene formation. PMA-treated basophils did not produce LTC4 in response to C5a. Rather, PMA blocked the IL-3 effect on C5a-induced LTC4 synthesis. Only the C5a signal but not the IL-3 effect was pertussis toxin sensitive. Two unrelated tyrosine kinase inhibitors, tyrphostin RG-50864 and herbimycin A, were both very efficient blockers of IL-3-dependent lipid mediator formation whereas C5a-induced histamine release was preserved. Thus LTC4 formation does not require activation of a staurosporine-sensitive serine/threonine kinase. To the contrary, IL-3-dependent LTC4 formation appears to be regulated by serine/threonine and tyrosine phosphorylation in an antagonistic manner.


Antigen presentation for T cell interleukin-2 secretion is a late acquisition of neonatal B cells.

The ability of B lymphocytes to process and present antigen to helper T cells is essential to initiate T cell-B cell interactions in humoral immune responses. Here we describe the developmental acquisition of the antigen-presenting function of B cells as measured by the ability of B cells to stimulate a T cell hybrid to interleukin (IL)-2 secretion. Neonatal splenic B cells are not adult-like in their ability to process and present the model protein antigen pigeon cytochrome (Pc), which enters the B cell through fluid-phase pinocytosis, until 21 to 28 days of life. The ability of neonatal B cells to process and present antigen which enters the cell bound to surface Ig is not adult-like until 28 days of age. When neonatal B cells acquire antigen-presenting cell (APC) function, surface IgM facilitates antigen processing. The delayed acquisition of APC function cannot be accounted for solely by a deficiency in major histocompatibility complex MHC class II, ICAM-1, or LFA-1 as neonatal B cells express adult levels of these molecules by 7-14 days after birth. Moreover, the ability of neonatal B cells to present a peptide fragment of Pc which does not require processing is adult like by day 14. Furthermore, neonatal B cells are capable of binding, internalizing and degrading radiolabeled antigen, suggesting a more subtle level of regulation. In contrast to neonatal B cells, immature B cells in the adult bone marrow and adult B cells undergoing antigen-driven differentiation to memory B cells, as defined by the loss of the J11D marker, are competent to process and present antigen resulting in T cell IL-2 secretion. Thus, developing B cell subpopulations in the adult and in the neonate can be distinguished. Only neonatal B cells are deficient in their ability to stimulate T cells to IL-2 production.


Expression and role of interleukin-2 receptor beta chain on CD4-CD8- T cell receptor alpha beta+ cells [corrected].

Using anti-murine interleukin-2 receptor beta chain (IL-2R beta) monoclonal antibody (mAb), we have examined the expression of IL-2R beta on murine thymocyte subpopulations. We found that it was constitutively expressed on 1%-4% of thymocytes in an almost mutually exclusive fashion with IL-2R alpha. The expression of IL-2R beta is developmentally regulated. While it is expressed mainly on T cell receptor gamma delta+ (TcR gamma delta+) cells during fetal age, the major subpopulation expressing IL-2R beta in adult mouse shifts to CD4-CD8-TcR alpha beta+ thymocytes. A considerable portion of CD4-CD8- TcR alpha beta+ cells in other organs, including spleen, bone marrow and liver, was also found to express IL-2R beta. In fetal thymus organ culture, the above thymocyte subset was induced to expand in response to exogeneous IL-2, and the expansion was inhibited by addition of anti-IL-2R beta mAb, suggesting that IL-2R beta is functional in this subpopulation. However, in vivo blockade of the IL-2/IL-2R pathway with the mAb did not exert any effects on the appearance of CD4-CD8- TcR alpha beta+ cells both in the thymus and the periphery. This indicates that the development of CD4-CD8- TcR alpha beta+ cells is not solely controlled by IL-2 but also by other complex elements.


Cloning and sequence analysis of two isotypic IgM heavy chain genes from Atlantic salmon, Salmo salar L.

A leukocyte cDNA library from Atlantic salmon, based on oligo-dT priming, was constructed in lambda-gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane-bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane-bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2%, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi-tetraploid state of the Atlantic salmon genome.


Expression of interleukin (IL)-1 beta, IL-6 and their respective receptors in the normal rat brain and after injury.

The expression of interleukin (IL)-1 beta, IL-6 and their respective receptors has been studied in the rat brain before and up to 24 h after injury. Messenger RNA transcripts of these four genes were detected by in situ hybridization (ISH) in different structures of the intact brain. The distribution was very similar for IL-1 beta, IL-6 and IL-6 receptor (IL-6R). The expression of IL-1R was more widespread. Within hours after injury, an increased expression of IL-1 beta, and thereafter of IL-6 was documented. The expression of IL-1R and IL-6R was also increased. This expression was bilateral and not restricted to the injured area. Within 24 h, all ISH patterns had returned to normal. The molecular data were confirmed by protein data. Indeed, the distribution of IL-6 (detected by immunocytochemistry) agreed with the ISH patterns for IL-6. Furthermore, extracellular fluid was collected by microdialysis at the site of the lesion during 12 h and successive fractions were assayed for the presence of bioactive IL-1 and IL-6. Increases in IL-1 and later in IL-6 levels were detected. The rapid and concomitant increased expression of IL-1 beta, IL-6 and their receptors after injury stresses their possible early role in inflammatory mechanisms also in the brain, before any recruitment of inflammatory cells from remote nervous and not nervous areas.


Cloning and sequences of primate CD4 molecules: diversity of the cellular receptor for simian immunodeficiency virus/human immunodeficiency virus.

To study the interaction between the primate lentiviruses simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) and the CD4 receptor we have cloned and sequenced the CD4 molecule from six non-human primate species: African green monkeys (three subspecies: sabeus, pytherethrus, aethiops), sooty mangabeys, patas monkeys, chimpanzees, rhesus macaques, and pig-tail macaques. Molecular cDNA clones representing CD4 mRNA were generated from total RNA from peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) amplification including reverse transcriptase in initial reactions followed by two rounds of nested amplifications. Primer sequences were selected from regions conserved among human and rodent CD4 genes. Alignments of deduced amino acid sequences revealed interesting findings. First, all of the primate CD4 molecules were about 90% identical to the human CD4 sequence except the chimpanzee (98%). Second, two macaques or two African green monkey subspecies were as distanly related as the human versus chimpanzee sequences. Third, relatedness of CD4 sequences could not be predicted on the basis of geographic origin (Asian vs. African). Finally, upon sequencing several clones from individual monkeys, a low degree of sequence variation (nucleotide substitutions, deletions, and insertions) was found within the same animal, and in case of sooty mangabeys two distict populations of CD4 molecules were present within three of four individuals. The distinguishing features involved eight amino acid changes, including a single lysine deletion relative to a primate consensus sequence in the first complementary-determing region of V1J1. These two CD4 populations were present also at the genomic DNA level and may arrive from the two chromosomal alleles, suggesting the existence of distinct sooty mangabey subspecies. Overall, the V1J1 and to a lesser extent V2J2 were the most variable regions among the sequences examined. By construction and expression in mammalian cell lines of CD4 chimeras in which these regions of the human CD4 were replaced by those of the African green monkey and pig-tail macaques, a higher molecular mass of the CD4 chimeras were obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the additional N-linked glycosylation sites present in these monkey CD4 are also used.


Heat shock prevents lipopolysaccharide-induced tumor necrosis factor-alpha synthesis by rat mononuclear phagocytes.

Tumor necrosis factor-alpha (TNF-alpha), a mononuclear phagocyte-derived peptide is known to participate in the pathogenesis of fever. To determine whether a feedback mechanism exists by which elevated temperatures influence TNF-alpha generation, we have examined the effects of heat shock on the in vitro synthesis of TNF-alpha by rat glomeruli, inflammatory peritoneal macrophages and blood monocytes. Preexposure of peritoneal macrophages to elevated temperatures for 20 min decreased the subsequent lipopolysaccharide-induced release of TNF-alpha bioactivity. The mean reductions were 11.9 +/- 5.0%, 86.3 +/- 12.0%, and 95.2 +/- 3.5% after pretreatment at 39, 41 and 43 degrees C, respectively. Reductions, that were transient, were maximum when lipopolysaccharide was added 0-2 h after heat shock. They correlated with the decreased release of immunoreactive TNF-alpha and the decreased expression of both cell-associated TNF-alpha molecule and TNF-alpha mRNA. Heat shock-induced inhibition of TNF-alpha release was independent of variations of prostaglandin synthesis, but was possibly related to the induction of heat-shock proteins since (a) macrophages exposed to heat shock synthesized the major 70- and 90-kDa heat-shock proteins, and (b) chemical inducers of the heat-shock response were also effective inhibitors of TNF-alpha release. The mean reduction of TNF-alpha release after pretreatment at 41 degrees C was found to be identical in glomerular tissue (82.0 +/- 7.5%), but significantly less in blood monocytes (43.9 +/- 10.9%). This supports the hypothesis that a negative-feedback mechanism exists between elevated temperature and lipopolysaccharide-induced TNF-alpha synthesis, and suggests that this regulation is less active in blood monocytes than in tissue macrophages.


Altered major histocompatibility complex-restricted antigen recognition by T cells from elderly humans.

Positive selection of T cells within the thymus gland leads to major histocompatibility complex (MHC)-restricted recognition of antigen by T lymphocytes. As the thymus gland involutes with age, altered MHC-restricted antigen recognition by T cells from elderly humans would be expected. We have tested this hypothesis by comparing the proliferative response of T cells and T cell clones from aged and young subjects to influenza determinants presented by autologous or allogeneic antigen-presenting cells (APC). Under conditions in which the allogeneic mixed lymphocyte reaction was minimal, T cells from six of seven aged donors but only one of seven young donors were stimulated by influenza vaccine presented by allogeneic APC. More importantly, one-half of the influenza-specific T cell clones derived from aged donors, but none of the clones derived from young donors, were activated by influenza vaccine presented by allogeneic APC. While 80% of the MHC-nonrestricted influenza-specific T cell clones expressed the gamma/delta T cell receptor, 20% of these clones expressed the alpha/beta T cell receptor. Thus, changes in MHC-restricted antigen recognition by T cells and in altered distribution of alpha/beta versus the gamma/delta T cell receptor bearing antigen-specific T cell clones occur with aging.


Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in the C57BL/6 immune response to NP.

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM- antigen-specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipopolysaccharide, interleukin (IL)-2, IL-4 and IL-5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti-NP antibody-secreting cells and non-secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody-secreting, IgG1+ cells were uniquely identified by co-expression of the matrix receptor, syndecan. The NP-specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ lambda+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.


Mechanisms of induction of primary virus-specific cytotoxic T lymphocyte responses.

We have investigated the ability of various antigen-presenting cell (APC) types to induce primary anti-viral cytotoxic T lymphocyte (CTL) responses by single in vitro stimulation. Of these APC types, only dendritic cells (DC) and RMA-S lymphoma cells could induce primary CTL responses, but by divergent mechanisms. DC were capable of generating primary virus-specific CTL, either by presenting viral peptide or processed infectious virus. In contrast, RMA-S cells could not present endogenous antigen, e.g. after virus infection, but this cell line very efficiently presented exogenous viral peptides to induce primary virus-specific CTL in vitro. Spleen cells, lipopolysaccharide-induced B cell blasts or the non-mutated RMA cells did not have the ability to trigger unprimed T cells by single in vitro stimulation. We have investigated several characteristics important for primary CTL response induction by DC and RMA-S cells (summarized in Fig. 6). Primary CTL response induction by DC or RMA-S cells was blocked by anti-LFA-1 or anti-CD8 monoclonal antibodies (mAb). DC rapidly aggregated with unprimed T cells, which was independent of LFA-1 and CD8 molecules. RMA-S cells did not form conjugates with unprimed T cells. Despite their abundant major histocompatibility complex (MHC) class I cell-surface expression, DC did not bind much exogenously added viral peptide. In contrast, the MHC class I molecules on RMA-S cells bound a large quantity of exogenously administered peptide. Powerful adhesion by DC and high expression of relevant MHC/peptide complexes on RMA-S cells are important features in the initial contact with unprimed T lymphocytes. In a later stage of contact, both DC and RMA-S cells activate LFA-1 (and CD8) molecules at the T cell surface to strengthen and maintain the contact between T cell and APC.


Selective and differential binding of interleukin (IL)-1 alpha, IL-1 beta, IL-2 and IL-6 to glycosaminoglycans.

The binding of interleukin (IL)-1 alpha, IL-1 beta, IL-2 and IL-6 to acidic polysaccharides was investigated by affinity chromatography of the recombinant, radioiodinated interleukins on columns of immobilized polysaccharide. Each interleukin showed selective binding retention. Overall heparin bound all four interleukins significantly, whereas chondroitin sulfate provided little retention. IL-1 alpha and IL-1 beta showed differential binding, with only the latter binding to hyaluronic acid. IL-2 was virtually completely retained on fucoidan. Noniodinated recombinant IL-2 bound similarly to fucoidan, and fucoidan was found to sequester IL-2 activity in a bioassay employing IL-2-dependent CTLL cells. In all other cases tested, interleukin retention was partial, implying that interleukin binding sites are sparsely distributed along the polysaccharide chains. These findings suggest that during the immune response, interleukins will tend to be retained at sites of secretion by interaction with glycosaminoglycans in the extracellular matrix and on cell surfaces.


Alpha heavy chain disease alpha mRNA contain nucleotide sequences of unknown origins.

Human alpha heavy chain disease is characterized by the production of abnormally short alpha IgH chains. In previously published cases it has been found that the malignant cells produce abnormal alpha mRNA, lacking VH and CH1 sequences and composed of a leader sequence peptide, sequences of variable length (69 to 84 bp) and of unknown origin, followed by normal CH2 and CH3 sequences. In this study we established the nucleotide sequence of alpha mRNA for six cases of alpha heavy chain disease. We observed that all six alpha mRNA lack the VH and CH1 sequences as do those previously described. They also contain in-frame inserts of unknown origin between the leader peptide and the normal CH2 and CH3 coding sequences. These inserts are of variable length (42 to 105 bp) and they are unrelated. These results suggest the existence of a common mechanism defect leading to deletions/insertions in alpha heavy chain disease rather than a specific interaction between alpha 1 IgH gene with a unique defined molecular species.


Effects of acute and chronic fenfluramine on self-stimulation and its facilitation by amphetamine.

DL-Fenfluramine (20 mg/kg) releasing serotonin and amphetamine (2 mg/kg) releasing dopamine were given to adult rats trained to bar press for electrical stimulation to the medial forebrain bundle. Amphetamine treatment enhanced lever-pressing for 1-2 h. A single fenfluramine treatment rapidly suppressed self-stimulation with slow recovery in 5-7 days to a rate below the initial basal rate. A second treatment a week later again suppressed response rate and rates returned to a still lower baseline. Combined fenfluramine-amphetamine treatment at this time transiently abolished lever pressing for 1-3 h followed by 9-11 h of enhanced responding. The serotonin antagonist, ketanserine (0.1 mg/kg), but not cyproheptadine (0.1 mg/kg i.p.), partially protected against the effects of fenfluramine. The serotonin agonist, quipazine (0.5 mg/kg), but not dimethoxyiodophenylisopropylamine (DOI) (2.2 mg/kg), partially substituted for fenfluramine in the combined treatment. Fenfluramine markedly depleted serotonin and 5-hydroxyindoleacetic acid in frontal cortex, hippocampus, and caudate putamen. Ventral and midline midbrain regions were less affected. Combined fenfluramine and amphetamine treatment elevated dopamine levels in frontal cortex, hippocampus and caudate-putamen, but not in midbrain. These findings support a serotonin-dopamine interaction in self-stimulation behavior and suggest that repeated fenfluramine treatment results in chronic low level serotonergic stimulation and diminished serotonin storage capacity.


The novel 5-HT1A receptor antagonist (S)-UH-301 prevents (R)-8-OH-DPAT-induced decrease in interstitial concentrations of serotonin in the rat hippocampus.

Previous studies have demonstrated that the novel 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) analogue (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin ((S)-UH-301) is able to antagonize several behavioural and biochemical effects of the 5-HT1A receptor agonist 8-OH-DPAT in the rat. In the present study in vivo microdialysis was used to evaluate the effects of (S)-UH-301 on interstitial concentrations of 5-hydroxytryptamine (5-HT), its metabolite 5-hydroxyindoloacetic acid (5-HIAA), and the catecholamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the dorsal hippocampus of freely moving rats. Furthermore, the effects of (S)-UH-301 on (R)-8-OH-DPAT-induced changes in dialysate hippocampal concentrations of 5-HT and metabolites were examined. Neither 5-HT nor metabolites were significantly influenced by (S)-UH-301 (1.25, 2.5, 5.0 mg/kg s.c.). In contrast, (R)-8-OH-DPAT (100 micrograms/kg s.c.) decreased interstitial concentrations of 5-HT (to 45% of baseline) and 5-HIAA (to 75%), and increased concentrations of DOPAC (to 165%) and HVA (to 155%). Pretreatment with (S)-UH-301 (2.5 mg/kg s.c.) 20 min before (R)-8-OH-DPAT (100 micrograms/kg s.c.) abolished the 5-HT and metabolite response to (R)-8-OH-DPAT. These data indicate that (S)-UH-301 is able to antagonize (R)-8-OH-DPAT-induced biochemical effects in vivo without producing any effects when given alone. Thus, the present study contributes to the characterization of (S)-UH-301 as a 5-HT1A receptor antagonist with low intrinsic activity.


Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G.

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy.


5-HT3 receptor antagonist effects of DAT-582, (R) enantiomer of AS-5370.

The serotonin 5-HT3 receptor antagonist effects of DAT-582, the (R) enantiomer of AS-5370 ((+/-)-N-[1-methyl-4-(3-methyl-benzyl)hexahydro-1H-1,4-diazepin-6- yl]-1H- indazole-3-carboxamide dihydrochloride), and its antipode were compared with those of AS-5370 and existing 5-HT3 receptor antagonists. In anesthetized rats, DAT-582 antagonized 2-methyl-5-HT-induced bradycardia with an ED50 value of 0.25 microgram/kg i.v., whereas the (S) enantiomer was without effect even at 1000 micrograms/kg i.v. In antagonizing the bradycardia, DAT-582 was as potent as granisetron, slightly more potent than AS-5370, and 2, 5 and 18 times more potent than ondansetron, ICS 205-903 and renzapride, respectively, although it was less potent than zacopride. DAT-582 inhibited cisplatin (10 mg/kg i.v.)-induced emesis in ferrets with an ED50 value of 3.2 micrograms/kg i.v. twice. The antiemetic activity of DAT-582 was more potent than that of the existing 5-HT3 receptor antagonists examined, except zacopride. In contrast, the (S) enantiomer had little effect at 1000 micrograms/kg i.v. twice. In isolated guinea-pig ileum, DAT-582 inhibited 5-HT-induced contractions with an IC50 value of 91 nM, whereas the (S) enantiomer hardly inhibited them even at 1000 nM. These results suggest that DAT-582, the (R) enantiomer of AS-5370, potently and selectively blocks 5-HT3 receptors.


Trans-ACPD induces a slow post-stimulus inward tail current (IADP) in guinea-pig olfactory cortex neurones in vitro.

The metabotropic glutamate receptor agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) produced a slow, persistent excitation of guinea-pig olfactory cortical neurones in vitro, and the appearance of a prominent post-stimulus after depolarization. The corresponding slow inward tail current (IADP) revealed under voltage clamp was insensitive to tetrodotoxin (or atropine) but was blocked by Cd2+ or tetrabutylammonium. The IADP properties resembled those of the slow inward tail current induced by muscarinic agonists in these neurones, suggesting a common intracellular transduction mechanism.


Mediation of thyrotropin-releasing hormone induced gastric motility increases in developing rats.

Centrally administered thyrotropin-releasing hormone (TRH) induces vagally mediated gastrointestinal effects which may be cholinergic, serotonergic or a combination. This study investigated mediation of TRH-stimulated gastric motility in developing rats. A serotonin (5-HT) antagonist (5-HT2, ketanserin or xylamidine; 5-HT3, MDL 72222) or an acetylcholine receptor blocker (atropine) was administered intraperitoneally 30 min prior to intracisternal TRH (5-10 micrograms). The 5-HT-depleting para-chlorophenylalanine (p-CPA) was administered 48 or 72 h prior to TRH. Gastric motility, monitored via extraluminal strain gauge, was not increased with TRH in atropine-pretreated rats. MDL 72222 had a significant age-related effect on TRH-induced gastric motility increases while 5-HT2 antagonists and p-CPA treatment did not. Thus, acetylcholine receptor blockade inhibits TRH-stimulated gastric motility in young and adult rats while 5-HT3 antagonism eliminates the motility response in young (7 and 10 days) rats.


Antinociceptive effects of Ca2+ channel blockers.

The antinociceptive action of four Ca2+ channel blockers, nifedipine, nimodipine, verapamil and diltiazem, was evaluated and compared to that of morphine using three algesiometric tests in mice and rats, namely, formalin, writhing and modified hot-plate test. Dose-response curves for all the drugs tested were similar and a significant dose-dependent antinociceptive action was evident in the formalin and writhing tests. However, in the hot-plate test, only nimodipine exhibited a significant analgesic effect, confirming the misleading results previously reported for this test. The findings suggest a pharmacological role of Ca2+ channel blockers in the modulation of antinociception under acute conditions. The analgesic action of Ca2+ channel blockers could be mediated by an increase in the nociceptive threshold resulting from interference with Ca2+ influx at opioid receptors, because Ca2+ influx is critical for the release of neurotransmitters and other substances implicated in nociception and inflammation. It is suggested that if a substance has a Ca2+ channel blocking effect, it should probably have some antinociceptive properties.


Possible mechanism of immunosuppressive effect of scoparone (6,7-dimethoxycoumarin).

The possible mechanism of the immunosuppressive effect of scoparone (6,7-dimethoxycoumarin) was investigated. Human peripheral blood mononuclear cells (10(6) cells/ml) were stimulated with 0.25% phytohemagglutinin (PHA) and the proliferative response was determined from the uptake of tritiated thymidine. Scoparone (10(-6) to 3 x 10(-4) M) reduced the proliferative response in a dose-dependent manner. The proliferative response of mononuclear cells to mixed lymphocyte reaction was also reduced by scoparone (10(-5) to 10(-4) M). Interleukin-1, interleukin-2 production and interleukin-2 receptor expression were all reduced in the presence of scoparone. Scoparone (10 and 30 microM) significantly reduced the suppression elicited by the diabetogenic drug, alloxan (10 mM). The suppressive activity of scoparone was significantly reduced by quinacrine (a phospholipase A2 inhibitor), indomethacin (a cyclooxygenase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor). The levels of prostaglandin E2, prostaglandin F2 alpha, leukotriene B4 and 2,3-dinor-thromboxane B2 in culture medium of PHA-stimulated mononuclear cells, measured with an enzyme immunoassay, were elevated by scoparone treatment. We compared the effect of scoparone on the mononuclear cell response to genistein, a specific inhibitor of protein tyrosine kinase and demonstrated the non-additivity and cross-desensitization of the two compounds. Our results suggest that the immunosuppressive effect of scoparone may be exerted in part through inhibition of protein tyrosine kinase and release of arachidonic acid metabolites.


1,3-Di(2-[5-3H]tolyl)guanidine labels more than one site in rat forebrain.

Studies of 1,3-di-(2-[5-3H]tolyl)guanidine ([3H]DTG) binding to rat brain membranes revealed that [3H]DTG binds to a high and a low affinity site with Kd values of 19.8 nM and 1.31 microM (corresponding Bmax values 291 fmol/mg protein and 8.68 pmol/mg protein). The order of potency of competitors for [3H]DTG binding revealed a binding profile typical of sigma site ligands. Several sigma ligands such as the enantiomers of 3-PPP (3-(3-hydroxyphenyl)-N- (n-propyl)piperidine) and (+/-)-pentazocine exhibited biphasic competition profiles for [3H]DTG binding, whereas other sigma ligands such as haloperidol displayed monotonic competition curves. Neither phenytoin nor carbamazepine were observed to enhance [3H]DTG binding. These data support the hypothesis that multiple sigma binding sites exist. The lack of phenytoin and carbamazepine modulation of [3H]DTG binding are in agreement with the proposed greater density of sigma site 2 in the rat, since allosteric modulation has been ascribed to the DM1/sigma 1 site.


Discriminative stimulus effects of 8-OH-DPAT in pigeons: antagonism studies with the putative 5-HT1A receptor antagonists BMY 7378 and NAN-190.

Pigeons were trained to discriminate 0.3 mg/kg of the 5-HT1A receptor agonist 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) from saline. RU 24969 (5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole), at doses of 5.6-10 mg/kg, and eltoprazine (5.6 mg/kg), both mixed 5-HT1A/B agonists, substituted completely for 8-OH-DPAT, whereas 3.0-10 mg/kg of the 5-HT1B/C agonist TFMPP (1-(m-trifluromethylphenyl)piperazine) and 0.1-3.0 of the 5-HT3 antagonist MDL 72222 (3-tropanyl-3,5-dichlorobenzoate) yielded only saline-appropriate responses. Substitution for 8-OH-DPAT by eltoprazine and RU 24969, which does not occur in rats, provides in vivo support for the suggestion that the absence of a 5-HT1B receptor in the pigeon allows more complete expression of 5-HT1A-mediated effects. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)]8-azaspirol-[4.5]- decane-7,9-dione) attenuated the 8-OH-DPAT stimulus at doses from 1.0 to 10 mg/kg but, when administered alone, also resulted in approximately 40% 8-OH-DPAT-appropriate responding at the highest dose. NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalamido)butyl)-piperazine (0.3-3.0 mg/kg) produced a dose-dependent and complete antagonism of the 8-OH-DPAT-discriminative stimulus; administered alone NAN-190 resulted only in saline-key responding. NAN-190 also reversed the rate-decreasing effects of higher doses of 8-OH-DPAT. The beta-adrenoceptor antagonist (+/-)-pindolol (5.6-17 mg/kg) antagonized the discriminative stimulus effects of lower 8-OH-DPAT doses but was unable to block the effects of higher doses of 8-OH-DPAT. Prazosin (1.0-10 mg/kg), which like NAN-190, is an alpha 1-antagonist, neither substituted for nor blocked the discriminative stimulus effects of 8-OH-DPAT. These results suggest that NAN-190 is an effective 5-HT1A receptor antagonist in this procedure with pigeons, with no indication of agonist actions, whereas BMY 7378 and pindolol are best characterized as partial 5-HT1A receptor agonists.


Characterization and autoradiographic distribution of [3H]AF-DX 384 binding to putative muscarinic M2 receptors in the rat brain.

The novel radioligand [3H]AF-DX 384 binds specifically and saturably to putative muscarinic M2 receptor sites in homogenates of rat cerebral cortex. In saturation studies, [3H]AF-DX 384 appears to bind to two subpopulations of sites/states, one of high affinity (Kd1 = 0.28 +/- 0.08 nM) and another of low affinity (Kd2 = 28.0 +/- 5.0 nM). The maximal binding capacity (Bmax) of [3H]AF-DX 384 binding sites represented 9.7 +/- 2.3 fmol/mg protein (Bmax1) and 1993 +/- 551 fmol/mg protein (Bmax2) for the high and low affinity sites/states, respectively. The ligand selectivity profile of [3H]AF-DX 384 (at 2 nM) revealed that (-)-quinuclidinyl benzylate = atropine greater than 4-diphenylacetoxy-N-methylpiperidine methobromide greater than AQ-RA 741 greater than AF-DX 384 greater than UH-AH 371 much greater than methoctramine greater than oxotremorine-M greater than hexahydro-sila-defenidol much greater than pirenzepine greater than carbamylcholine much much greater than nicotine. This suggests that under our assay conditions [3H]AF-DX 384 binds mostly to M2-like muscarinic receptors in the rat central nervous system. This is further supported by the clear M2-like pattern of distribution observed using quantitative receptor autoradiography. High densities of specific labelling were seen in areas such as the hypoglossal nucleus, the pontine nucleus, the superior colliculus, the motor trigeminal nucleus, various thalamic nuclei and certain cortical laminae. Compared to [3H]AF-DX 116, the percentage of specific binding detected with [3H]AF-DX 384 was much higher. This is likely to be related to the greater chemical stability and affinity of [3H]AF-EX 384. In addition, autoradiograms obtained with [3H]AF-DX 384 (2 nM) are of better quality with film exposure periods five shorter than those needed for [3H]AF-DX 116 (10 nM). Therefore, [3H]AF-DX 384 displays a good selectivity for muscarinic M2 sites and offers major advantages, including higher affinity and greater stability, over previously used ligands.


Blockade of hyperalgesia and neurogenic oedema by topical application of nitroglycerin.

Surprisingly, a single topical application of a nitroglycerin (NTG) gel in humans has been shown to cause analgesia and to reduce oedema in thrombophlebitis. In the present investigation, we showed that the NTG gel reduces prostaglandin E2-induced hyperalgesia and blocks neurogenic inflammation induced in rat skin by antidromic electrical stimulation of the saphenous nerve. These results offer an explanation for the effects of topical application of NTG observed in thrombophlebitis, which may be common to other cutaneous pathologies. The data also support the development of nitrates the effects of which are restricted to the site of application.


Neurotoxic effect of prenatal exposure to MPTP on the dopaminergic systems of the marmoset brain.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was incidentally administered to pregnant marmosets during the whole gestational period, except for the last 15 days before term. The infant monkeys were killed 5 months after birth, and dopamine and its metabolites were measured in the striatum and the nucleus accumbens. Prenatal exposure to MPTP produced a marked dopamine depletion in these brain regions of the offspring, showing that MPTP is able to cross the placental barrier in primates.


Isoflurane anesthesia is stereoselective.

The anesthetic effects of the (+) and (-) isomers of isoflurane were determined in mice. Like the clinically important racemate, both isomers produced dose-dependent increases in anesthetic sleep time. A statistically significant (P less than 0.005, ANOVA) difference in the potencies of these isomers ((+) greater than (-)) was observed. To our knowledge, this is the first demonstration of a stereospecific action of a volatile anesthetic in vivo.


Influence of rhein anthrone and rhein on small intestine transit rate in rats: evidence of prostaglandin mediation.

The present study was undertaken to investigate the role of prostaglandins in the shortening of transit time observed after intraduodenal administration of rhein anthrone and rhein. After intraduodenal administration of rhein anthrone (0.5-10 mg/rat), a dose-dependent acceleration of small intestinal transit was observed. The effect for rhein (1-10 mg/rat) was far less pronounced. In the same test conditions, analysis of small intestinal tissue revealed a significant increase of prostaglandin E2 (PGE2), reaching its maximum value 30 min after administration of rhein anthrone. The increase in PGE2 found 30 min after administration of rhein was not significant. The effects provoked by rhein anthrone could be largely prevented by pretreatment of the animals with indomethacin (1-3 mg rat) or cortisol (10 mg/rat). It is concluded that prostaglandins play an important role in the acceleration of the transit provoked in rats by rhein anthrone.


The developmental electrophysiologic effects of moricizine HCl on the canine cardiac Purkinje fiber.

Using standard microelectrode techniques, the developmental cellular electrophysiologic effects of moricizine HCl on adult and neonatal canine Purkinje fibers were studied. Steady state and rate-related changes in the transmembrane action potentials produced by moricizine HCl in both age groups were characterized and compared. Also, the rate of barium-induced abnormal automaticity before and after drug was also investigated in neonatal and adult Purkinje fibers. The major findings of this study are as follows. (1) The steady state and rate-related depressant effects of moricizine HCl on Vmax were similar in both age groups. (2) Moricizine HCl shortened APD90 in the adult fibers to a greater extent than in the neonate. (3) The concentration of moricizine HCl required to significantly reduce the rate of abnormal automaticity was less in the neonate than in the adult. The effect of moricizine HCl on APD90 of individual Purkinje fibers is influenced both by their control APD90 value as well as by maturational factors. It is less clear whether developmental differences in the effects of moricizine HCl on abnormal automaticity are solely a result of differences in control rates of abnormal automaticity between the two age groups.


Effects of YM435, a novel dopamine D1 receptor agonist, on pancreatic exocrine secretion in anesthetized dogs.

The effects of YM435, a novel dopamine (DA) D1 receptor agonist, on pancreatic exocrine secretion were investigated in anesthetized dogs. Each drug was injected i.a. as a single bolus. Graded doses of YM435 (0.3-30 nmol) produced dose-dependent increases in the rate of secretion of pancreatic juice, with a maximum effect at approximately 10 nmol, and with a high concentration of bicarbonate and low concentration of protein. SCH23390 (3-30 nmol), a selective D1 receptor antagonist, caused a progressive parallel shift to the right of the dose-response curve for YM435-stimulated pancreatic secretion without changing the maximum response. Schild analysis of the data indicated that the inhibitory constant (Ki) value was 2.9 nmol, and that SCH23390 inhibited YM435-stimulated pancreatic secretion in a competitive manner. Both DA (0.01-3 mumol) and SKF38393 (0.3-30 mumol), a selective D1 receptor agonist, also increased the secretory rate and bicarbonate concentration, and decreased the protein concentration to the same extent as YM435. These results suggest that YM435 is a potent stimulant of pancreatic exocrine secretion by acting on DA D1 receptors of the pancreas in dogs.


Effect of kynurenate on functional deficits resulting from traumatic spinal cord injury.

The potential role of excitatory amino acid (EAA) receptors in spinal cord trauma was examined in a standardized rat model of contusive injury. EAA antagonists were administered in a split-dose protocol with half given 5 min prior to and the remainder 15 min after contusion produced at the T8 vertebral level. Hindlimb function was assessed using a battery of tests of reflex and more complex behaviors at 1 day after injury and weekly thereafter through 4-8 weeks. Functional deficits were compared for groups administered intravenous MK 801 (1 mg/kg), dextromethorphan (10 mg/kg) and kynurenate (300 mg/kg) or the vehicle, saline, alone. In addition, possible effect of the drugs themselves on hindlimb function were assessed in uninjured controls. None of the drugs produced more than transient effect on uninjured rats. In contused rats, only kynurenate produced significant reductions in functional deficits as compared to saline controls. Significant improvement of hindlimb function was also observed when the thoracic cord was locally perfused with kynurenate via intrathecal cannulas and when kynurenate was directly infused into the contusive injury site by stereotaxic microinjection. Using the latter route of administration, a dose-dependent effect of kynurenate (100, 200 and 400 nmol) on the ability of contused rats to use their hindlimbs in locomotion was demonstrated. The highest dose also resulted in a significant reduction in overall functional deficits from 1 week through 1 month and at 2 months after injury. Our results support the hypothesis that EAA receptors at or near the injury site are involved in producing a proportion of the overall functional deficits stemming from traumatic injury.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of semotiadil fumarate (SD-3211) on renal hemodynamics and function in dogs.

Studies were carried out to define the effect of semotiadil on renal hemodynamics, renal function and renin release in pentobarbital-anesthetized dogs. Intrarenal arterial infusion of semotiadil resulted in a significant increase in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow, urinary excretion of electrolytes and renin release. Semotiadil did not affect the linear relationship between osmolar clearance and the free water reabsorption rate, and increased the urinary excretion of sodium and calcium to the same extent. These results suggest that the main tubular site of action of semotiadil is the proximal tubule. Intrarenal infusion of a potent non-peptide angiotensin II antagonist, DuP753 (15 micrograms/kg per min), resulted in an increase in RBF, GFR, urine flow and UNaV. In spite of the blockade of the intrarenal renin angiotensin system(RAS) with DuP753, semotiadil caused almost the same effects as it did in the absence of DuP753. These results suggest that the renal effects of semotiadil are independent of the intrarenal RAS.


The influence of changes in perfusion pressure and angiotensin II on the renal excretory responses to atrial natriuretic peptides.

The renal actions of atriopeptin III were examined in anaesthetised rats at differing perfusion pressures before and following blockade of the renin-angiotensin system. At normal perfusion pressure 1000 ng kg-1 atriopeptin III caused reversible increases in glomerular filtration rate, of 20%, urine flow, absolute and fractional sodium excretions of 51-93%. Reduction of left renal perfusion pressure to 80 mm Hg decreased glomerular filtration rate by 30% and urine flow, absolute and fractional sodium excretions by 80% while atriopeptin III administration only minimally changed these variables. Concomitantly, right kidney perfusion pressure rose by 15 mm Hg which significantly increased fluid output, while the atriopeptin III induced diuresis and natriuresis were significantly larger. During infusion of captopril 900 micrograms kg-1 h-1 when pressures at the left and right kidneys had been reduced and elevated, respectively, atriopeptin III caused larger excretory responses in both kidneys which were greater than without captopril. These result suggested that the atriopeptin III mediated natriuresis and diuresis were directly proportional to perfusion pressure and attenuated by angiotensin II.


Differences between vasorelaxant responses of the canine and human mesenteric arteries and veins to amrinone.

Amrinone induced similar degrees of relaxation in human mesenteric arteries and veins contracted with KCl, but exerted a more potent vasodilator effect in canine mesenteric veins than in arteries. Dibutyryl cAMP had a stronger effect on KCl-induced contraction in canine mesenteric veins than in arteries. These results suggest that amrinone promotes a different vasodilating action in human and canine mesenteric vessels, possibly via different sensitivity to cAMP in each vascular smooth muscle.


Autoradiographic evidence for the occlusion of rat brain dopamine D3 receptors in vivo.

[125I]Iodosulpride binding was studied in frontal rat brain sections by quantitative autoradiography. Using preincubated (= washed) sections, selective labelling and identification of dopamine D3 receptors was obtained using 0.2 nM [125I]iodosulpride in the presence of 100 nM domperidone for the occlusion of the D2 receptors. A high density of D3 receptors was noticed in the islands of Calleja. When preincubation of the sections was omitted, no D3 receptor labelling could be achieved, indicating tight binding to the receptor of an endogenous inhibitor. Such a tight receptor occupancy was not observed for the D2 receptor and various other neurotransmitter receptors. The occlusion of the D3 receptor could be prevented by tetrabenazine-induced monoamine depletion of the rats. It can be concluded, therefore, that D3 receptors are massively occupied by a monoamine, likely to be dopamine. This observation prompts the question to what extent dopamine D3 receptors can become occupied in vivo by systematically applied exogenous compounds.


Antiulcer and gastric secretory activity of N-phthaloyl gamma-aminobutyric acid.

N-Phthaloyl gamma-aminobutyric acid, a new gamma-aminobutyric acid derivative synthesized in this laboratory, has been found to possess anticonvulsant, antinociceptive and antistress activities. Effects of this derivative on gastric lesions induced by aspirin and ethanol were studied in rats. N-Phthaloyl gamma-aminobutyric acid significantly inhibited both aspirin and alcohol ulceration. The ED50 in each case being 76.34 and 43.65 mg/kg i.p. respectively. The volume of gastric acid secretion was diminished but gastric mucus secretion was significantly enhanced. The antiulcer effect was blocked by bicuculline and 3-mercaptopropionic acid. We conclude that (a) N-phthaloyl gamma-aminobutyric acid possesses antiulcer activity (b) the new derivative is probably a non-specific gamma-aminobutyric acid receptor agonist (c) the observed activity may be due to a mucoprotective action.


O-acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase alpha-smooth muscle actin expression in cultured arterial smooth muscle cells.

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC50 = 2 and 1 micrograms/ml respectively, versus 31 micrograms/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of alpha-smooth muscle actin and alpha-smooth muscle actin mRNA.


Effects of oxodipine on isolated rabbit aorta and mesenteric resistance vessels.

The inhibitory effects of the dihydropyridine Ca2+ antagonist, oxodipine, on contractions and 45Ca2+ influx stimulated by noradrenaline (NA) and high K+ in rabbit aorta were compared to the same parameters measured in mesenteric resistance arteries. In aortic rings oxodipine, 10(-11)-10(-6) M, inhibited in a concentration-dependent manner the contractions induced by high K+ (IC50 = 9.0 +/- 4.0 x 10(-10) M) or by Ca2+ in high K+ solution (IC50 = 6.2 +/- 2.4 x 10(-9) M), while responses to NA were only slightly affected (IC50 greater than 10(-6) M). In mesenteric resistance vessels oxodipine inhibited the contractions induced by high K+ and NA but was more effective against NA- than high K(+)-induced contractions (IC50 = 5.2 +/- 3.1 x 10(-10) and 1.2 +/- 1.8 x 10(-8) M, respectively). The concentration-inhibition curves for high K(+)-induced contraction and 45Ca2+ influx in aorta were almost superimposable (I50 = 2.2 +/- 2.0 x 10(-9) M), whereas NA-induced contractions were inhibited less than 45Ca2+ influx (I50 = 8.2 +/- 2.6 x 10(-8) M). In mesenteric resistance vessels the curves for contraction and 45Ca2+ influx stimulated by high K+ and NA were also superimposable, but 45Ca2+ influx stimulated by NA was more sensitive to oxodipine than that stimulated by high K+ (I50 = 3.9 +/- 2.0 x 10(-10) and 2.2 +/- 1.2 x 10(-8) M, respectively). It is concluded that the effects of oxodipine can be attributed to its ability to inhibit Ca2+ entry through both potential- and receptor-operated pathways.(ABSTRACT TRUNCATED AT 250 WORDS)


Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.


Endothelins inhibit serotonin-induced platelet aggregation via a mechanism involving protein kinase C.

Endothelins are a family of three peptides that act as local hormones released by the endothelium. They were found to inhibit rabbit and dog platelet aggregation in vivo, but no effect was observed in vitro. In order to investigate the possible interaction between endothelins and human platelet serotonin receptors, their effects on platelet aggregation induced by serotonin was studied. Endothelin-1, -2 and -3 had a dual action, on platelet aggregation and calcium mobilization induced by serotonin. When added at the same time as serotonin, endothelin potentiated the response to the amine. On the contrary, preincubation of platelet suspension with endothelin resulted in a concentration-dependent inhibition of the serotonin-mediated platelet response. Moreover, endothelin-1 inhibited serotonergic amplification of epinephrine-induced aggregation of platelets. We hypothesize that endothelins can bind to the platelet membrane and interact with serotonin receptors. The diverse effect of endothelins on serotonin-induced aggregation and calcium mobilization may be due to stimulation of protein kinase C.


Further characterization of the 5-hydroxytryptamine 5-HT1-like receptor mediating contraction of guinea-pig iliac artery.

We recently described a 5-hydroxytryptamine 5-HT1-like receptor mediating contraction in guinea-pig isolated iliac artery. The present study was aimed at characterizing this receptor with respect to the currently recognized 5-HT1 receptor subtypes (5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D). The potencies of 13 drugs tested as agonists correlated with their affinities for 5-HT1D binding sites only. The concentration-response curve for 5-carboxamidotryptamine (5-CT, a 5-HT1-like receptor agonist) was unaffected by propranolol (10 microM), which is reported to have affinity for 5-HT1A, 5-HT1B and 5-HT1C recognition sites. Yohimbine (3 microM) and metergoline (1 microM) antagonized 5-CT with pKB values of 6.15 and 6.96, respectively. These values are close to those found in a functional correlate of 5-HT1D sites in the same species, namely the presynaptic 5-HT autoreceptor in guinea-pig brain cortex. The overall results support the view that the receptor studied is of the 5-HT1D subtype. The receptor shares close similarities with other vascular 5-HT1-like receptors mediating contraction, for example the receptor present in dog saphenous vein.


Selective chiral inhibition of Ca2+ entry promoted by bisbenzyltetrahydroisoquinolines in rat uterus.

The effects of diltiazem and six bisbenzyltetrahydroisoquinoline alkaloids (antioquine, 7-O-methylantioquine, dimethylantioquine, monterine, granjine and cordobimine) were studied in rat isolated uterus in order to clarify the mechanisms of their relaxant actions. All the compounds tested completely relaxed KCl-induced contractions and totally or partially inhibited oxytocin-induced rhythmic contractions. Only alkaloids with absolute configurations (1R,1'S or 1R,1'R) acted intracellularly, promoting relaxation of contractile responses induced by oxytocin in a Ca(2+)-free medium, as does papaverine. Alkaloids of the antioquine series (1S,1'R) selectively inhibited Ca2+ entry. The great rigidity of these structures and their stereoselective action make these alkaloids useful in studies of the conformational features of the Ca2+ channel.


The angiotensin hexapeptide 3-8 fragment potently inhibits [125I]angiotensin II binding to non-AT1 or -AT2 recognition sites in bovine adrenal cortex.

In the present studies, ligand competition experiments were conducted to examine the ability of angiotensin II peptide agonists and nonpeptide AT1- and AT2-selective receptor antagonists to inhibit the binding of [125I]angiotensin II to bovine adrenal cortical membranes. Angiotensin II, angiotensin III, the All-(3-8) hexapeptide fragment of angiotensin II, and the AT1-selective receptor antagonist L-158,809, inhibited [125I]angiotensin II binding in a biphasic fashion indicative of a ligand interaction at more than one recognition site. Approximately 20% of low affinity [125I]angiotensin II binding was inhibited only by high micromolar concentrations of L-158,809. RG 13647 (1(-1,4-benzodioxan-2-methyl)-5-diphenylacetyl-4,5,6,7-tetra hydro-1H-imidazo- [4,5,c]-pyridine-6-carboxylic acid) represents a potent and AT2-selective analog of PD 123177 and showed weak activity in competing for [125I]angiotensin II binding with an IC50 value of 100 microM. When subsequent competition studies were conducted in the presence of 1 microM L-158,809 to block [125I]angiotensin II to the AT1 receptor subtype, the angiotensin II agonists produced monophasic inhibition curves with AII-(3-8) showing the greatest activity (IC50 = 6 nM) followed by angiotensin III (IC50 = 15 nM) much greater than angiotensin II (IC50 = 110 nM). RG 13647 was not found to significantly inhibit this portion of [125I]angiotensin II binding. These data demonstrate that bovine adrenal cortex contains both the AT1 receptor subtype, as well as, a novel class of [125I]angiotensin II recognition sites which may be analogous to the recently described angiotensin IV (AT4) receptor.


JMV 449: a pseudopeptide analogue of neurotensin-(8-13) with highly potent and long-lasting hypothermic and analgesic effects in the mouse.

We recently reported that H-Lys psi (CH2NH)Lys-Pro-Tyr-Ile-Leu-OH (JMV 449), a pseudopeptide analogue of neurotensin-(8-13) with a reduced CH2NH bond in position 8-9, was about 3 times more potent than neurotensin in binding to mouse brain membranes and in contracting the guinea-pig ileum, and was markedly more resistant to degradation than neurotensin when exposed to rat brain membranes. In the present study, we compared the time courses and dose-response relationships for the ability of i.c.v. injected neurotensin and JMV 449 to elicit hypothermia and analgesia (tail-flick test) in the mouse. The results show that the pseudopeptide analogue behaved as a highly potent and long-lasting neurotensin agonist in these two in vivo bioassays. The analogue should prove very useful for studying the effects of chronic neurotensin receptor stimulation in vitro and in vivo.


Effect of LY 83583 on relaxation induced by non-adrenergic non-cholinergic nerve stimulation and exogenous nitric oxide in the rat gastric fundus.

In the rat gastric fundus, the reported cGMP-lowering agent LY 83583 (10(-5) M) inhibited the relaxation induced by nitric oxide (NO), without altering the response to isoprenaline, vasoactive intestinal polypeptide, sodium nitroprusside or electrical field stimulation of inhibitory non-adrenergic non-cholinergic neurones, which are thought to release NO. Incubation with superoxide dismutase partially prevented the effect of LY 83583. When added during a relaxation maintained by continuous NO infusion, LY 83583 reversed the relaxation. It is concluded that LY 83583 inactivates exogenous NO through the generation of superoxide anions.


Tissue-specific localization of mitochondrial imidazoline-guanidinium receptive sites.

In the present report, we studied the distribution of the imidazoline-guanidinium receptive site in mitochondrial fractions from different rabbit and human tissues. Binding studies of the imidazoline-guanidinium receptive site ligand [3H]idazoxan, allowed to distinguish two groups of tissues: the first one, including kidney, brain and liver, displays a high density of imidazoline-guanidinium receptive site; the second one, consisting of striated and smooth muscle, enterocytes, lung, spleen and heart, is characterized by 4- to 16-fold lower binding site density. The demonstration that mitochondrial imidazoline-guanidinium receptive sites are not equally expressed in all tissues can be considered as a further progress towards the characterization of their functional activity.


Endothelin-1 enhances vascular permeability in the rat heart through the ETA receptor.

Intravenous injection of endothelin-1 (ET-1, 0.1 and 1 nmol/kg) resulted in a dose-dependent increase in vascular permeability in the coronary circulation of conscious rats. The increase was almost completely abolished by the selective ETA receptor antagonist, BQ-123 (1 mg/kg). The ET-1 analogue, [Trp(For)21]ET-1, which is devoid of the depressor but not the pressor activity, evoked changes in protein extravasation similar to those with ET-1. These data indicate that the permeability effect of ET-1 is mediated through the ETA receptor.


Cocaine place preference is blocked by the delta-opioid receptor antagonist, naltrindole.

Naltrindole, a selective delta-opioid receptor antagonist, was evaluated for its potential to block the reinforcing properties of cocaine using a conditioned place pairing paradigm in Lewis rats. Cocaine HCl (15 mg/kg s.c.) produced a strong place preference which was significantly blocked in animals pretreated with naltrindole (3 mg/kg i.p.); naltrindole alone showed no reinforcing or aversive effects. The results suggest a novel approach for the treatment of cocaine abuse in man.


Cross tolerance between cromakalim and RP49356 in the uterus of the rat in vivo and in vitro.

We investigated tolerance to the potassium (K+) channel openers, cromakalim and RP49356 and cross tolerance between them in the uterus of the rat. Continuous infusion of RP49356 in vivo produced 5.2-fold (200 or 300 micrograms/kg per h) and 7.2-fold (600 micrograms/kg per h) desensitization of the uterus to RP49356. Uterine sensitivity to RP49356 was reduced 7.4-fold in cromakalim-tolerant rats and sensitivity to cromakalim was reduced 7.9-fold in RP49356-tolerant rats. Incubation of isolated uterus with 10 microM cromakalim for 4 h reduced uterine sensitivity to cromakalim by about 45-fold and to RP49356 by 2.1-fold but did not alter sensitivity to salbutamol. Incubation with 10 microM RP49356 for 4 h reduced sensitivity to RP49356 by 2.5-fold and to cromakalim by 3.8-fold. Occurrence of uterine tolerance to K+ channel openers in vitro suggests that tolerance in vivo has a pharmacodynamic basis. Cross tolerance between cromakalim and RP49356 provides evidence that these two K+ channel openers possess common binding sites and/or mechanism(s) of action. However, the greater extent of self-tolerance to cromakalim than to RP49356 suggests a difference in their actions.


Intestinal relaxation by endothelin isopeptides: involvement of Ca(2+)-activated K+ channels.

Our previous studies have shown that endothelin-1 (ET-1) induces an initial relaxation followed by a contraction in the guinea-pig ileum. To test whether other ET isopeptides (ET-2, ET-3, vasoactive intestinal contractor (VIC) and sarafotoxin S6b) and big ET-1, the ET-1 precursor, also induce similar biphasic responses, we compared their effects in isolated guinea-pig ileum. In addition, the mechanism of initial relaxation was studied. At 1-100 nM, ET-1, ET-2 and VIC were equipotent in producing the biphasic responses. S6b also produced similar biphasic responses, except that only a relaxation was elicited at 1 nM. ET-3 was approximately 30- to 100-fold less active than ET-1 in producing the contraction, whereas it was as potent as ET-1 in producing relaxation. Big ET-1 induced a relaxation of slower onset and longer duration, followed by a weak contraction at concentrations higher than 30 nM. The initial relaxation produced by ET-1 was not affected by pretreatment with L-NAME (NW-nitro-L-arginine methyl ester), hemoglobin, 9-AC (anthracene-9-carboxylic acid), SITS (4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid), glibenclamide, ouabain, phorbol 12,13-dibutyrate, sodium nitroprusside, human atrial natriuretic peptide (hANP) or forskolin, whereas it was abolished by pretreatment with apamin. Although phorbol 12,13-dibutyrate pretreatment had no significant effect on the biphasic response of ET-1, it rapidly reversed the sustained contraction produced by ET-1. These results indicate that the initial relaxation is caused by the activation of Ca(2+)-activated K+ channels.


Effects of chronic treatment with caffeine on kidney responses to angiotensin II.

Previous studies demonstrate that chronic administration of caffeine causes glomerular filtration to deteriorate in rats with high-renin renovascular hypertension. A partial explanation for these findings could be that chronic administration of caffeine alters the effects of angiotensin II on the kidney. As an initial test of this hypothesis, we compared the acute effects of intrarenal infusions of angiotensin II (3 ng/min) on renal function in control rats versus rats treated with 0.1% caffeine in their drinking water for 1 week. The renal responses to angiotensin II in a group of animals receiving acute intrarenal infusions of adenosine (10 micrograms/min) were also measured to determine whether caffeine and adenosine modulated renal responses to angiotensin II in opposite directions. All studies were performed in the in situ blood perfused rat kidney. Neither caffeine nor adenosine significantly altered angiotensin II-induced changes in renal blood flow, urinary excretory function or renin release. However, caffeine augmented and adenosine attenuated the increase in filtration fraction caused by angiotensin II. The fact that caffeine potentiates angiotensin II-induced increases in filtration fraction without affecting angiotensin II-induced reductions in renal blood flow is consistent with, but does not prove, the hypothesis that chronic administration of caffeine modifies the effects of angiotensin II on the renal microvasculature. If this inference is correct, caffeine could facilitate renal damage in high-renin hypertension by exacerbating angiotensin II-induced increases in glomerular capillary hydrostatic pressure.


Blood pH and the actions of nifedipine on cardiac excitation-contraction coupling.

Hearts from male Sprague-Dawley rats were perfused, by the Lagendorff method, with calcium-free Krebs solution (containing either adrenaline or a high level of potassium) at pH 7.48 (control), 7.26 (acidosis) or 7.69 (alkalosis). When the hearts stopped contracting, a dose of nifedipine or its vehicle was given before measuring the force of contraction, coronary perfusion pressure and heart rate in response to graded doses of calcium. The calcium-antagonising efficacy of nifedipine was reduced during acidosis in both adrenaline- and potassium-stimulated hearts, but the reduction was greater in the adrenaline-stimulated hearts. Alkalosis led to a small increase in the efficacy of nifedipine on adrenaline- and potassium-stimulated contractions.


Na+,2Cl-,K+ cotransport system as a marker of antihypertensive activity of new torasemide derivatives.

A series of compounds related to torasemide, a loop diuretic, were synthesized and examined for their diuretic potency and inhibitory activity on the erythrocyte and renal medullary thick ascending limb vesicle Na+,2Cl-,K+ cotransport in Milan hypertensive (MHS) and normotensive (MNS) rat strains, where previous studies had demonstrated an alteration of the cotransport system genetically related to hypertension. From the results of the screening, structure-activity relationships were drawn and two compounds, JDL 961 and C 2921 were selected. Their IC50 on renal vesicle cotransport were similar in the two strains (JDL 961: MHS = 1.8 microM; MNS = 1.2 microM; C 2921: MHS = 4 microM; MNS = 3.8 microM), and were 4-8 times lower than those of torasemide (MHS = 13 microM; MNS = 31 microM, P less than 0.01) and 50-60 times lower than those of bumetanide (MHS = 145 microM; MNS = 206 microM, P less than 0.05) taken as reference compounds. Their ability to reduce the development rate of hypertension was tested both in MHS and in Okamoto spontaneously hypertensive rats (SHR) strain, in which cotransport alterations are opposite to those of MHS. Both torasemide derivatives (7.5 mg.kg-1 os per day) prevented development of hypertension in the two strains. The time course of this hypotensive activity was faster and the percentage of blood pressure fall greater in MHS (20-25%) than in SHR rats (12-15%), even though the absolute value of blood pressure fall was similar in MHS (JDL 961 = -17 mm Hg; C 2921 = -30 mm Hg) and SHR (JDL 961 = -25 mm Hg; C 2921 = -20 mm Hg). A superimposable effect of bumetanide was observed in the two strains, but at 8 times higher daily dose (60 mg.kg-1). These results suggest that new loop diuretics can be selected for their antihypertensive activity on the basis of their in vitro potency in inhibiting the Na+,2Cl-,K+.


Different utilization of Ca2+ in the contractile action of endothelin-1 on cerebral, coronary and mesenteric arteries of the dog.

Vasoconstrictor responses to endothelin-1 (ET) were compared between endothelium-denuded strips of cerebral, coronary and mesenteric arteries of the dog. Contractile responses to lower concentrations (below 3 x 10(-10) M) of ET were significantly greater in the cerebral and coronary arteries than in the mesenteric artery. The cerebral and coronary arteries, but not the mesenteric artery, relaxed significantly from the resting level when placed in a 0-Ca solution. Readdition of Ca2+ to the cerebral and coronary arteries placed in the 0-Ca solution caused a biphasic contraction which was susceptible to inhibition by nifedipine. When ET below 10(-10) M was introduced before the Ca2+ contraction, this peptide produced no detectable contraction, but augmented the Ca2+ contraction. The augmented Ca2+ contractions were abolished by 10(-7) M nifedipine. These effects of ET were not observed in the mesenteric artery. The contractile responses of the mesenteric artery to ET determined in the presence of elevated extracellular K+ concentrations were comparable to the responses of the cerebral artery to this peptide determined in the presence of normal K+ concentrations. These results indicate that the enhanced responses to ET in the cerebral and coronary arteries were dependent on the Ca2+ influx through voltage-dependent Ca2+ channels and suggest that these channels are in an activated state when these arteries are in a resting state.


Antihypertensive and vasodilator effect of A-80b, a new pyridazino indole derivative.

The hypotensive and antihypertensive activities of a A-80b, a newly synthesized pyridazino[4,5-b]indole derivate were investigated in anaesthetized rats. In vitro studies were also done to examine the possible mechanism of its vasodilator action. A 80b (3-15 mg/kg i.p.) showed potent and long-lasting antihypertensive activity in spontaneous hypertensive rats. In normotensive rats, A-80b (7.5-30 mg/kg i.p.) also lowered blood pressure but less than in hypertensive rats. The decrease in diastolic pressure was greater than the decrease in systolic pressure and cardiac frequency was not modified significantly. Contractile responses induced in isolated rat thoracic aorta by K+ and noradrenaline were inhibited by A-80b. In K(+)-depolarized rat aorta, A-80b showed dose-dependent inhibition of the Ca(2+)-induced contraction. Also, A-80b inhibited spontaneous contractions of rat portal vein. The vasodilator action seemed to be endothelium-independent. These results suggest that A-80b is a new chemical entity which exerts a hypotensive and antihypertensive effect, possibly attributable to vasodilator activity via interference with Ca2+ influx and probably Ca2+ mobilization from intracellular stores.


The spinal and peripheral roles of bradykinin and prostaglandins in nociceptive processing in the rat.

The effect of peripheral and intrathecal administration of the bradykinin B2 receptor antagonist HOE140 and the non-steroidal anti-inflammatory drug indomethacin were studied on the response of dorsal horn nociceptive neurones to formalin in the anaesthetized rat. Peripheral pretreatment with HOE140 reduced both phases of the formalin response whereas subcutaneous administration of indomethacin (5 mg/kg) reduced only the second phase. Pre-treatment with intrathecal HOE140 resulted in a dose-dependent reduction of the second phase whereas a high dose (500 micrograms) intrathecal indomethacin reduced both phases of the response. Bradykinin plays a role in peripheral nociception during the first and second phase of the response whereas the prostaglandins are only involved during the second phase. The converse is true centrally, the prostaglandins appear to be involved in both phases of the formalin response whereas bradykinin only plays a central role during the second phase of the response.


Memantine prevents quinolinic acid-induced hippocampal damage.

Memantine, used as a drug for treatment of spasticity and other extrapyramidal disorders as well as dementia, was shown to prevent brain damage caused by the glutamate (N-methyl-D-aspartate, NMDA) receptor agonist, quinolinic acid. Studies were focused on the hippocampal formation which is known to be highly vulnerable to quinolinate. Pretreatment of animals with memantine added to the food led to a reliable protection of hippocampal neurons when the drug was administered chronically for a period of 10 days prior to quinolinate exposure (i.c.v. injected). Additional i.p. administration of memantine (simultaneously with quinolinic acid or up to 24 h later) did not substantially add to the protective potency of the memantine diet. Our findings indicate that memantine may have beneficial effects in the treatment of brain disorders which are mediated by excitotoxic effects of glutamate.


Heterogeneity of contractile 5-HT receptors in human hand veins.

To increase our knowledge of human peripheral vasospasm we characterized the contractile 5-hydroxytryptamine (5-HT) receptors in human superficial hand vein segments in vitro. The 5-HT1 receptor agonist, sumatriptan, the 5-HT2 receptor agonist, dl-alpha-methyl-5-HT, and the 5-HT3 receptor agonist, 2-methyl-5-HT, all induced concentration-dependent contractions. The contractile response to sumatriptan was antagonized by the non-selective 5-HT receptor antagonist, methiothepin, but was unaffected by the 5-HT2 receptor antagonist, ketanserin. The contractile response to dl-alpha-methyl-5-HT was antagonized by both methiothepin and ketanserin. The contraction elicited by 2-methyl-5-HT was not affected by the 5-HT3 receptor antagonist, MDL 72222, but was antagonized by ketanserin. The results suggest that serotonergic contraction in the human superficial hand vein involves both 5-HT1 and 5-HT2 but not 5-HT3 receptors. Such receptor heterogeneity in human blood vessels should be considered when using drugs and when designing future compounds for medical use.


Pregnenolone sulfate increases the convulsant potency of N-methyl-D-aspartate in mice.

The neurosteroid pregnenolone sulfate is known to specifically enhance NMDA-gated currents in spinal cord neurons. The response does not appear to be mediated by the glycine/NMDA modulatory site. Here we found that pregnenolone sulfate significantly increased the convulsant potency of N-methyl-D-aspartate (NMDA), but not of pentylenetetrazol (PTZ). In agreement with previous in vitro reports showing that the glutamergic NMDA receptor is also specifically modulated by steroids, our findings suggest that pregnenolone sulfate selectively activates the NMDA receptors involved in convulsions in the intact animal.


Olvanil: more potent than capsaicin at stimulating the efferent function of sensory nerves.

The capsaicin analogue olvanil stimulated an increase in cutaneous blood flow when injected intradermally into the anaesthetised rabbit, as measured using a 133Xenon clearance method. Olvanil was found to be a 10-fold more potent vasodilator (on a molar basis) than capsaicin. The effect of both vasodilators was significantly inhibited by the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37). These findings suggest that olvanil stimulates the efferent function of cutaneous sensory nerves in a more potent manner than capsaicin. We therefore suggest that olvanil is a useful pharmacological tool for studying the activity of neuropeptides released from sensory nerves.


AF102B, a muscarinic M1 receptor agonist, mimics some effects of acetylcholine on neurons of rat hippocampus slices.

The putative muscarinic M1 receptor agonist, AF102B, was applied to rat hippocampal slices and the responses of intracellularly recorded pyramidal cells were examined. AF102B mimicked some effects of acetylcholine on these cells as follows: at low concentration, AF102B attenuated a slow after-hyperpolarization in response to a long depolarizing current pulse. This effect was blocked by the M1 antagonist, pirenzepine. At higher concentrations, AF102B also depolarized the cells and caused an increase in their input resistance. AF102B did not affect local excitatory postsynaptic potentials or reactivity to topically applied excitatory amino acid substances. These experiments indicate that AF102B acts as an agonist at some muscarinic M1 receptor subtypes in mammalian brain.


The selective protein kinase C inhibitor, NPC 15437, induces specific deficits in memory retention in mice.

We studied the effects of a selective inhibitor of protein kinase C (PKC), 2,6-diamino-N-[(1-(1-oxotridecyl)-2-piperidinyl]methyl)hexamide (NPC 15437), on acquisition and memory retention of a Y-maze avoidance task in mice. Post-training administration of NPC 15437 (0.1-10 mg/kg i.p.) induced a dose-dependent deficit in retention of the temporal but not the spatial component of the task. This selective amnesia does not reflect state dependence and NPC 15437 (1 mg/kg) had no effect on acquisition and memory retrieval. Our results suggest that this new PKC inhibitor interferes with mechanisms underlying memory consolidation. This is in agreement with recent findings suggesting that PKC is involved in memory processes.


Direct vascular actions of hydrochlorothiazide and indapamide in isolated small vessels.

The mechanism by which thiazides lower peripheral resistance is unresolved. The aim of this study was to investigate the mechanisms of the acute vasodilator action of hydrochlorothiazide and the 'thiazide-like' diuretic indapamide on human, guinea pig and rat small arteries. Vessels were mounted on a myograph and the relaxation profile of both drugs and interactions with K+ channels and the vascular eicosanoid system were studied. Neither drug had any relaxant effect in rat mesenteric vessels and indapamide did not relax human arteries. Hydrochlorothiazide in both human and guinea pig vessels produced significantly more relaxation of noradrenaline than K(+)-constricted vessels (P less than 0.001). Relaxation to hydrochlorothiazide was reduced in the presence of charybdotoxin. Maximal-induced hydrochlorothiazide relaxation was reduced by 64% in human arteries (P less than 0.001) and by 91% in guinea pig vessels (P less than 0.001). Incubation with glibenclamide and indomethacin had no effect on the relaxant activity of hydrochlorothiazide and indapamide. Indapamide-induced relaxation was unaffected in the presence of charybdotoxin. These results show marked differences in the acute vasodilator action of hydrochlorothiazide and indapamide. There appears to be involvement of Ca(2+)-activated K+ channels in the acute vasorelaxant activity of hydrochlorothiazide.


5-Hydroxydecanoate inhibits ATP-sensitive K+ channel currents in guinea-pig single ventricular myocytes.

We investigated the effect of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the electrical activity of guinea-pig ventricular myocytes. The outward K+ current increased by lowering the intracellular ATP concentration (0.5 mM) was efficiently blocked by 5-hydroxydecanoate when recording in the whole cell configuration with the application of voltage ramps. The increase in the time-independent outward K+ current induced by reducing intracellular ATP to 0 mM was also blocked by 5-hydroxydecanoate (10 or 100 microM) and by tolbutamide (1 mM). Using the single channel recording technique, we found that 5-hydroxydecanoate blocked ATP-sensitive K+ channels when its channel open probability was increased by 1 mM ATP together with 1 mM ADP or by an intracellular pH of 6.6. These conditions are well documented to reflect metabolic changes in the early stages of myocardial ischemic attack. These results suggest that 5-hydroxydecanoate could inhibit ATP-sensitive K+ channels, resulting in an antiarrhythmic effect specifically on ischemic hearts.


Prostanoid-mediated vascular contraction in normotensive and hypertensive rats.

We investigated the role of prostanoids in the constrictor effect of calcium ionophore A23187, endothelin-1 and vasopressin in rings of thoracic aorta obtained from normotensive rats and rats with aortic coarctation-induced hypertension. Isometric tension was measured in aortic rings bathed in buffer with and without indomethacin (10 microM), CGS13080 (10 microM) or SQ29548 (1 microM) to inhibit cyclooxygenase and thromboxane synthase and to block TxA2-PGH2 receptors, respectively. Increases in tension elicited by A23187 and vasopressin in aortic rings from hypertensive rats exceeded responses in rings from normotensive rats. A23187-induced contractions were virtually abolished by indomethacin and SQ29548, and slightly attenuated by CGS13080. These agents also attenuated the contractions elicited by endothelin but not by vasopressin. According to these data, a prostanoid(s) agonist for TxA2-PGH2 receptors contributes to the constrictor effect of A23187 in aortic rings of hypertensive rats, and of endothelin in aortic rings of normotensive and hypertensive rats. Moreover, the expression of prostanoid-mediated contractions as it pertains to the aortic response to A23187 is greatly increased in hypertensive rats.


Homologous tachyphylaxis to bradykinin and its interference with allergic pleurisy in actively sensitized rats.

After recovery from a first intraplantar or intrathoracic stimulation with bradykinin, repeated daily provocation with this peptide resulted in a progressive loss of its ability to cause paw or pleural oedema, reaching 0-20% of the control within seven and four consecutive provocations, respectively. The phenomenon was shown to be time reversible, since the unresponsiveness ceased when stimulations were discontinued, and localized, since paw oedema evoked by the peptide was not modified after desensitization of either the contralateral paw or the pleural cavity. Furthermore desensitization to bradykinin did not influence the pleurisy elicited by either histamine (200 micrograms/cavity), 5-hydroxytryptamine (5-HT) (100 microgram/cavity) or platelet-activating factor (PAF) (1 microgram/cavity), suggesting that the desensitization was selective. In contrast, when actively sensitized animals were submitted to bradykinin-induced tachyphylaxis, pleural exudation and leukocyte influx induced by antigen were drastically reduced, strongly implying bradykinin in this process. We demonstrated that repeated daily stimulation with bradykinin cause selective, local and reversible auto-refractoriness, which may be useful as a tool in attempting to evaluate the role of this peptide in inflammation.


Doxorubicin: an antagonist of muscarinic receptors in guinea pig heart.

While studying the mechanisms of doxorubicin-induced cardiotoxicity, we observed that doxorubicin inhibited the negative inotropic effect of acetylcholine in isolated heart muscle preparations. We therefore examined the effects of doxorubicin on muscarinic acetylcholine receptors. In left atrial muscle preparations isolated from guinea pig heart and stimulated at 2 Hz at 30 degrees C, doxorubicin caused a parallel right-ward shift of the dose-response curves for the negative inotropic effects of acetylcholine. The inhibitory action was reversed by an additional incubation in the absence of doxorubicin. Doxorubicin reversed the carbachol-induced inhibition of developed tension: a high concentration of doxorubicin brought the force back to its original strength. Doxorubicin inhibited specific [3H]quinuclidinyl benzilate (QNB) binding to membrane preparations obtained from ventricular muscle of guinea pig hearts. The pA2 value for doxorubicin obtained in the inotropic study corresponded to the IC50 value for doxorubicin observed in the [3H]QNB binding assay. These results indicate that doxorubicin acts as a weak competitive antagonist on muscarinic acetylcholine receptors.


Cardiovascular effects of the new nitric oxide donor, pirsidomine. Hemodynamic profile and tolerance studies in anesthetized and conscious dogs.

The hemodynamic profile of pirsidomine, a new donor of NO (nitric oxide), was evaluated in dogs. In anesthetized dogs, the intravenous or intraduodenal administration of pirsidomine (0.3-10 mg/kg) decreased dose relatedly the preload and afterload of the heart, total peripheral resistance, cardiac output, left ventricular work and myocardial oxygen consumption. In conscious renal-hypertensive dogs, oral administration of pirsidomine (1.0-10 mg/kg) caused a marked and sustained decrease in systolic blood pressure and left ventricular end-diastolic pressure, which was accompanied by a slight and transient increase in heart rate and contractility. The diastolic blood pressure was affected less than in anesthetized dogs. Similar hemodynamic effects were obtained with M1 (3-(1-(2,6-dimethylpiperidino))-sydnonimine; 0.3-1 mg/kg), the main metabolite of pirsidomine, and with the known NO donor, isosorbide-5-mononitrate (IS-5-MN; 2-10 mg/kg). Tolerance development after repeated administration of pirsidomine and IS-5-MN was also investigated. In anesthetized dogs, repeated intraduodenal administrations of pirsidomine did not attenuate the response whereas tolerance occurred with hemodynamically equieffective doses of IS-5-MN. In conscious dogs, long term oral treatment, three times daily every 8th h for 5 days, revealed tolerance to IS-5-MN, slight or no tolerance to pirsidomine, and no cross-tolerance between the two agents. The results indicate that pirsidomine possesses an antianginal hemodynamic profile similar to that of its main metabolite, M1, and of IS-5-MN. This suggests a common mode of action via the release of NO.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of angiotensin II and angiotensin III on airway epithelial short-circuit current: involvement of pertussis toxin-sensitive G protein.

The effects of angiotensin II (AII) and angiotensin III (AIII) on bioelectric properties of canine cultured tracheal epithelium were investigated. Both peptides increased the short-circuit current (Isc), an effect that was accompanied by the release of prostaglandin (PG) E2 and was abolished by indomethacin and diphenylamine-2-carboxylate but not by amiloride. The AII action was not altered by amastatin. The increases in Isc induced by AII and AIII were inhibited by pertussis toxin, whereas cholera toxin had no effect. Thus, both peptides may selectively stimulate airway epithelial Cl- secretion through the activation of pertussis toxin-sensitive regulatory G protein and the subsequent generation of PGE2.