Kaito Tsurumi

Heterogeneity of circulating and exudated polymorphonuclear leukocytes in superoxide-generating response to cyclic AMP and cyclic AMP-elevating agents : investigation of the underlying mechanism

It has been found that cyclic AMP and cyclic AMP-elevating agents inhibit formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide production from polymorphonuclear leukocytes (PMNs). The quantitative differences of this inhibitory effect on human and rabbit blood versus human salivary and rabbit peritoneal (tissue) PMNs were investigated. PMNs from all sources showed the same pattern of fMLP-stimulated superoxide generation, although it was slightly higher in tissue PMNs. However, treatment with salbutamol differentially blunted fMLP-stimulated superoxide production from blood PMNs compared with tissue PMNs in both human and rabbit. While it could inhibit production from blood PMNs by 30-60%, it had only a negligible effect on generation from tissue PMNs. Similarly, forskolin, phosphodiesterase IV inhibitor Ro-201724, and dibutryl cyclic AMP showed significantly higher inhibitory effects on superoxide generation from blood PMNs than tissue PMNs in both species. beta-Adrenergic receptors, cyclic AMP accumulation, and protein kinase A activity were investigated in blood versus tissue PMNs to clarify the mechanism underlying the above-mentioned differences. At the beta-adrenergic receptor level, no significant changes were detected in the number or the binding affinity of the receptors in tissue versus blood PMNs of human and rabbit. On the other hand, cyclic AMP accumulation was significantly higher in response to salbutamol and Ro-201724 in fMLP-stimulated blood versus tissue PMNs in human and rabbit. At the same time, blood PMNs showed significantly higher cyclic AMP-dependent protein kinase A activity than tissue PMNs in human and rabbit. We concluded that tissue PMNs are less responsive to the effect of cyclic AMP-elevating agents in terms of fMLP-stimulated superoxide inhibition. This is due to differences, at least, at two levels. The first is lower accumulation of cyclic AMP and the second is lower protein kinase A activity in tissue versus blood PMNs.

Hypothermic prevention of the hippocampal damage following ischemia in mongolian gerbils comparison between intraischemic and brief postischemic hypothermia

The protective effect on ischemic hippocampal damage was compared between intra- and postischemic hypothermia in Mongolian gerbils and its regional preference was evaluated. Male Mongolian gerbils were subjected to transient forebrain ischemia and the hippocampus 7 days after ischemia was examined histologically. In the intraischemic hypothermia (29-31 degrees C) group, CA1 damage was completely prevented in spite of spontaneous postischemic hyperthermia. In contrast, the same degree of brief postischemic hypothermia exerted no preventive effect. While neurons in the subiculum and CA2 sector were also protected against ischemic damage by intraischemic hypothermia, injured pyramidal neurons were always seen in the CA4 sector.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2-200 microM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 trisphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

A proposal for purification of salivary polymorphonuclear leukocytes by combination of nylon mesh filtration and density-gradient method: A validation by superoxide- and cyclic AMP-generating responses

Isolation and purification of salivary polymorphonuclear leukocytes (SPMNs) from accompanying epithelial cells was presented by using a density-gradient method with Ficoll. SPMNs samples prepared by already established methods (nylon mesh filtration) was compared with SPMNs samples after further purification by Ficoll (d = 1.083). Microscopically, SPMNs samples after nylon mesh filtration contain higher percentage of epithelial cells than SPMNs samples after Ficoll centrifugation. In response to stimulation of superoxide generation, both samples showed the same pattern of response. However, in response to forskolin and prostaglandin E1, cyclic AMP levels in samples after nylon mesh purification were significantly higher than in samples after Ficoll purification because of the presence of contaminating epithelial cells. We can conclude that, although nylon mesh filtration is satisfactory when we need to examine superoxide generation but further purification is necessary when we want to measure factors like intracellular cyclic AMP formation.

Effect of opioids on delayed neuronal death in the gerbil hippocampus.

The effect of opioids on delayed neuronal death was evaluated in the gerbil hippocampus. Male Mongolian gerbils were subjected to transient forebrain ischemia and neuronal density was evaluated in the hippocampus 7 days following ischemia. When hypothermia during and after ischemia was prevented, treatment with morphine, U-50488H, or naloxone provided no significant protection. In contrast, a spontaneous drop in rectal temperature to 32 degrees C at the end of ischemia produced near-complete protection of CA1 pyramidal neurons. No opioids modulate the protective effect of hypothermia.

Glucagon modulates superoxide generation in human polymorphonuclear leucocytes

It has been found that leucocytes possess receptor sites for glucagon and glucagon was shown to increase during bacterial infection. To verify the interconnection between glucagon, leucocytes and bacterial infection we studied the effect of glucagon on superoxide generation and second messenger transduction in PMNs. We found that glucagon could not stimulate chemiluminescence by itself but it could enhance FMLP- but not PMA-induced chemiluminescence in a concentration (50-800 pg/ml) dependent manner. However, after incubation of PMNs with 10 microM of ST-638 (a tyrosine kinase inhibitor) the enhancement effect converted into inhibitory effect. We also found that glucagon treatment of PMNs increased both IP3 and cyclic AMP levels as second messengers. ST-638 greatly attenuated the IP3 increment in the glucagon-treated FMLP-stimulated PMNs. From these results we can conclude that glucagon could enhance superoxide generation from FMLP-stimulated PMNs by elevating IP3. Inhibition of IP3 increment by tyrosine kinase blockade uncover the inhibitory effect of the increasing cyclic AMP on superoxide production.

Dopaminergic Unique Affinity of Tetrahydroberberine and l-Tetrahydroberberine-d-Camphor Sulfonate

l-Tetrahydroberberine-d-camphor sulfonate (THB-CS) possessed an inhibitory effect on apomorphine-induced chewing movement in a similar manner to that of tetrahydroberberine (THB). Both compounds enhanced barbiturate-induced hypnosis. They did not have an anticonvulsant effect on convulsive seizures induced by bicuculline, pentetrazole or strychnine. THB and THB-CS blocked dopamine-stimulated adenylate cyclase activity. These compounds showed almost equipotent affinities to dopamine D1 (3H-SCH-23390) and D2 (3H-spiperone) receptors but did not have significant affinity to mu-opioid, muscarinic and alpha 2-adrenergic receptors, and benzodiazepine binding sites. Furthermore, both compounds did not elicit cataleptogenic behavior, even at very high doses. These data suggest that THB and THB-CS have a central depressant effect through both D1 and D2 dopaminergic receptors and may have different modes of action from that of standard neuroleptics.

Characterization of opioid receptors in the Mongolian gerbil cerebellum

Characteristics of opioid receptors binding in the Mongolian gerbil cerebellum were determined by in vitro radioligand binding technique. Homogenate of cerebellar membranes possessed a binding capacity for 3H-DAMGO, a mu-agonist, and for 3H-diprenorphine, an antagonist of mu, delta and kappa-receptors. These bindings were saturable, and characterized by high affinity (Kd values: 1.55 +/- 0.43 nM for 3H-DAMGO and 0.56 +/- 0.20 nM for 3H-diprenorphine) and high density (Bmax: 127.8 +/- 23.8 fmoles/mg protein for 3H-DAMGO and 135.8 +/- 9.03 fmoles/mg protein for 3H-diprenorphine). Autoradiographically, the binding sites were predominantly located in the molecular layer of the cerebellum. 3H-DPDPE, a delta-agonist, showed only very small binding capacity to the cerebellar membranes. The kappa-agonist, 3H-U-69593, also showed very small binding capacity to the cerebellar membranes except in the early postnatal period. Thus, the gerbil cerebellum can be considered as a tissue containing a homogeneous population of mu-opioid receptors.

Opioid and non-opioid binding of β-endorphin to bovine adrenal medullary membranes

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.

Opioid receptor binding in CXBK mice brain

It has been reported that the CXBK strain mouse is deficient in μ1 type opioid receptor. The properties of opioid receptor binding in CXBK were compared with those of C57BL/6, progenitor mouse strain of CXBK. In the membrane preparations from whole brain, CXBK demonstrated a small Bmax value for 3 H-DAGO binding and a large Kd value for 3 H-U-69593 binding, compared with those of C57BW6. In all brain regions of CXBK, except cerebellum, the 3 H-DAGO binding showed lower values. The CXBK strain exhibited a significant decrease in the 3 H-U-69593 and 3 H-DPDPE-Cl binding to striatum