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.

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.

Effects of cytosine arabinoside on rat and rabbit embryos cultured in vitro.

The technique of rabbit whole embryo culture for 48 or 24 hr from day 9, 10 or 11 of gestation has been improved for elucidation of species differences. The effects of 1-beta-d-arabinofuranosylcytosine (ara-C) and its metabolite 1-beta-d-arabinofuranosyluracil (ara-U) on cultured rat and rabbit embryos were examined. Slc:SD rats on day 10.5 of gestation were explanted and cultured in rat serum containing ara-C (5-10 mug/ml) for 48 hr. Rabbit embryos of the Japanese White strain on day 9 or day 10 of gestation were explanted and cultured in rabbit serum containing ara-C (0.03-1.0 or 3-30 mug/ml) or ara-U (1.0 or 30 mug/ml) for 48 or 24 hr. Cultured rat embryos exposed to ara-C showed abnormalities of the head (malformations of the telencephalon, mesencephalon and rhombencephalon), mandible and limb bud, and short tail. Growth parameters, such as crown-rump length, head length, protein content and somite number, were reduced with increasing concentrations of ara-C. In the rabbit, embryos cultured from day 9 of gestation for 48 hr showed abnormalities of the head (telencephalon, rhombencephalon), mandible and limb bud with ara-C at 0.1 mug/ml and higher concentrations. Concentration-dependent decreases in crown-rump length, head length and protein content were observed. The findings in embryos cultured from day 10 of gestation were similar to those in embryos cultured from day 9. Ara-U produced no detectable abnormalities in embryos cultured for 48 hr from day 9 of gestation, or for 24 hr from day 10. These results indicate that ara-C has teratogenicity in vitro that is similar in both rat and rabbit embryos.

[Interactions between 6-chloro-5-cyclohexyl-1-indancarboxylic acid (TAI-284) and biopolymers (author's transl)].

TAI-284, a new non-steroidal acidic analgesic and anti-inflammatory agent was investigated and the interactions with biopolymers were compared with those of indomethacin (IMC) and ibuprofen (IP). TAI-284 inhibited the heat denaturation of bovine serum albumin at pH 5.3, similar to that seen with IMC and weaker than that seen with phenylbutazone. TAI-284 prevented the rat erythrocyte from heat-induced hemolysis and was twice as potent as IMC. TAI-284 produced alterations in platelet function as characterized by loss of secondary aggregation in response to ADP and inhibition of aggregation by collagen. Both these effects were one fifth as potent as those seen with IMC. In rats, platelet aggregation induced by collagen and secondary ADP aggregation was reduced in a dose-dependent manner by a single oral administration of TAI-284. The inhibitory activity was approximately one fourth that of IMC or twice that of IP in the former and in the latter one fifth that of IMC or similar as that of IP. These results suggest that the essential feature of TAI-284 is its potent membrane stabilizing action which is considered to be an necessary mechanism in the action of anti-inflammatory drugs. It is considered that TAI-284 may be less active than IMC in inhibiting prostaglandin biosynthesis in platelets.

[Interaction of phenothiazinic anti-inflammatory agent, protizinic acid, with the biopolymers: Inhibitory effects on functions of platelets (author's transl)].

Two types of phenothiazinic anti-inflammatory agents, protizinic acid (PZA) and metiazinic acid (MZA) were examined using 1) heat denaturation test, 2) heat-induced erythrocyte lysis, 3) several platelet functions, 4) model membrane systems containing the same phospholipids and cholesterol compositions as in platelets. PZA and MZA were inhibited with heat denaturation in a similar manner seen with BSA and heat-induced erythrocyte lysis, and effects were more potent than indomethacin (IM). PZA showed inhibitory effects similar to MZA on ADP or collagen-induced platelet aggregation. However, in arachidonic acid (AA)-induced rabbit platelet aggregation, PZA had a more potent effect, similar to effects seen with IM and more potent than those of MZA. PZA inhibited the lethal effect of AA in rabbits at concentrations lower then MZA. To determine the sites of action, we examined the effects on uptake and release reactions of 3H-serotonin. PZA and MZA did not affect the uptake reaction but did reduce the release of serotonin to a greater extent than seen with IM. The tested drugs had little effect on the platelet aggregation in vivo. To investigate the interaction of these drugs with lipid bilayers, we used liposomes as a model membrane, of which the lipids compositions were the same as that of platelets. The tested drugs showed inhibitions of the liposome aggregation with addition of 6 mM Ca2+, in a dose dependent manner and similar to findings in the drug-platelet system. In this experiment, PZA had a more potent interaction with lipid bilayers than did MZA. These results suggest that interactions of PZA with the platelet membrane may be the origin of the PZA-induced inhibition of the platelet aggregation, in addition to the effect on the biosynthesis of prostaglandins.

A human skeleton from the Ohguruwa remains.

In 1941, the Ohguruwa remains were discovered at the Mizuho sports ground site in Nagoya. They date from about 3000 B.C., which is the early Jomon era. When the stadium was reconstructed in 1980, four human skeletons were found. Three of them, however, were in poor condition and moreover, were incomplete. However, the second skeleton was in good condition and could almost be reconstructed, and this skeleton (No. 2) was used for our study. It was found in the classic posture with arms and legs folded. Some pieces of a dog's skeleton were also discovered near the No. 2 skeleton's chest. This particular skeleton generally showed characteristics typical of the Jomon era. It had a stout structure and was judged to be a middle aged male because of the following features. There was considerable attrition of the occlusal surfaces on the remaining teeth. The teeth were worn flat, probably owing to the hard food and to their use as a tool. Both canines and first premolars of the upper and lower arches had been extracted in accordance with the custom of the time. Typical caries and localized periodontal breakdown were not observed, although there was horizontal alveolar bone loss, especially in the anterior regions. The mastoid process was extremely large and prominent. The lateral prominence of the mandible was developed. Analysis of lateral cephalogram revealed that the mandible was in the anterior position. The angles of SNA, SNB and ANB were 89.6 degrees, 89.2 degrees and 0.4 degrees, respectively. The skeletal pattern was definitive Class III. The adaptive changes in the teeth, their supporting tissues, temporomandibular joints and the related muscles--the harmonious masticatory system--were all estimated.