The protease-activated receptor (PAR) is the family of G protein-coupled, seven transmembrane domain receptors, currently consisting of four members, PARs 1-4. The activation of PARs occurs by proteolytic unmasking of the N-terminal cryptic receptor-activating tethered ligand. In the past decade since the cloning of PAR-1, physiological roles that PARs play have been gradually understood and are now considered extremely extensive and important. This review describes physiological and/or pathophysiological roles of PARs in the circulatory, digestive, respiratory and central nervous systems, on the basis of our works and of those achieved by other research groups. The future perspective of studies on PARs is also discussed, focusing on the possibility of clinical application of PAR-targeted drugs.
Interleukin 1 (IL-1) is one of the inflammatory cytokines, which plays a pivotal role in both host defense and homeostasis. Its signal is transduced by type I IL-1 receptor (IL-1RI). This report gives an insight into the regulatory mechanism of IL-1RI in both in vitro and in vivo. IL-1 up-regulates IL-1RI through prostaglandin E2 (PGE2) production on human fibroblasts. However, in the presence of indomethacin, IL-1 down-regulates the receptor by destabilizing IL-1 receptor mRNA. Type I and type II interferons (IFNs) up-regulate the expression of IL-1RI. This up-regulation leads to the increasing susceptibility of IL-1RI to IL-1, as the DNA binding of IL-1-induced NF-κB and the production of IL-1-induced IL-6 from the fibroblasts are augmented by pretreatment with IFNs. On the other hand, the expression of cell surface IL-1RI is inhibited by tyrosine kinase inhibitors, herbimycin and genistein, resulting in reduction of the kinase activity of IRAK (IL-1 receptor associated kinase) and IL-1-induced IL-6 production from the fibroblasts. Lipopolysaccharide (LPS) augments the expression of IL-1RI mRNA and cell surface molecule in the hepatocytes of mice in vivo, and the augmentation is mediated by the interaction of IL-1, IL-6, and of glucocorticoid (GC). When hepatocytes were pretreated with dexamethasone (Dex) and IL-6, the activation of IRAK was augmented in response to IL-1, indicating that IL-1 signaling is also up-regulated. In addition, IL-1 treatment ather combined administration of Dex and IL-6 into mice markedly increased the serum level of serum amyloid A. These data suggest that the expression of IL-1RI is regulated by inflammatory cytokines, PGE2, GC and LPS in vitro and in vivo. This study shows that the biological activity of IL-1 can be controlled by regulating the expression of IL-1RI, and therefore proposes the use of pharmaceutical drugs for the regulation of cytokine expression.
Phospholipase A2 receptor (PLA2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family and mediates a variety of biological responses elicited by group IB secretory phospholipase A2 (sPLA2-IB). In the present study, we have shown the evidence that a novel type of sPLA2, sPLA2-X, also acts as one of the high-affinity ligands for mouse PLA2R. We then generated PLA2R-deficient mice and found that the knockout mice exhibited the resistance to an endotoxic shock with reduced plasma levels of proinflammatory cytokines, such as TNF-α and IL-1β. In situ hybridization analysis revealed that the expression of PLA2R transcript was markedly enhanced in type II alveolar epithelial cells and a subset of splenic lymphocytes in accordance with the elevated expression of sPLA2-IB and TNF-α mRNAs during endotoxic shock. In addition, the elevated expression level of TNF-α transcript was significantly reduced by the deficiency of PLA2R, suggesting that PLA2R plays a role in the regulation of TNF-α expression in these cell types. We then synthesized a specific sPLA2 inhibitor, indoxam, which blocked the binding of sPLA2-IB and X to PLA2R. Indoxam was found to suppress the elevation of the plasma level of TNF-α and prolonged the survival of endotoxin-challenged mice. The inhibitory effects of indoxam were abolished by the deficiency of PLA2R, demonstrating the involvement of PLA2R in the progression of endotoxic shock. We also detected and characterized a soluble form of PLA2R protein in the plasma of mouse with anti-PLA2R antibody, and showed its potential role as an endogenous sPLA2 inhibitor. Taken together, a series of studies with PLA2R-knockout mice have elucidated a critical role of PLA2R in the regulation of the development of endotoxic shock.
The physiological role of the prostaglandin (PG) E2 receptor EP4 subtype was investigated by the generation of EP4-deficient mice by gene targeting. Loss of the EP4 receptor was not lethal in utero, but most EP4 (−/−) neonates became pale and lethargic approximately 24 h after birth, and died within 72 h. Less than 5% of the EP4 (−/−) mice survived and grew normally more than a year. Marked congestion in the pulmonary capillaries were observed before death, suggesting that EP4 (−/−) neonates had left-sided heart failure. Histological examination revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization study showed that EP4 mRNA was strongly expressed in the ductus. The treatment of indomethacin, an inhibitor of PG synthesis, on wild-type fetus induced constriction of ductus arteriosus, while the ductus in EP4 (−/−) fetus was insensitive to indomethacin. These results suggest that neonatal death is at least partly due to patent ductus arteriosus, and that the EP4 receptor plays a role in the regulation of the patency of this vessel. They also indicate that the normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.
A novel method of calculating the water-accessible molecular surface area from the number of points generated on the molecular surface was developed. This method yielded a molecular surface area with high accuracy and speed. The molecular surface area of lecithin shows an excellent linear correlation with the logarithm of the critical micelle concentration for many lecithins having different acyl chains. The solution structure of oxyphenonium bromide estimated from the molecular surface area approach was close to that obtained from NMR. Furthermore, the change of molecular surface area, ΔS(HG), with docking of host and guest was defined and its calculation method was developed. Because both the host and the guest generally consist of hydrophilic and hydrophobic atomic groups, ΔS(HG) was divided into such four terms as ΔSoo(HG), ΔSow(HG), ΔSwo(HG), and ΔSww(HG). For instance, ΔSoo(HG) is the decrease in surface area with contact between the hydrophobic surfaces of the host and the guest. When the guest molecule was moved along the symmetry axis of cyclodextrin (CyD), the structure of a complex having the maximum value of ΔSoo(HG) corresponds with the crystal structure. The solution structures of several inclusion systems were predicted by this method. For various systems including α-CyD, β-CyD, γ-CyD, and aromatic and aliphatic guests, the maximum values of ΔSoo(HG) showed a good correlation with the logarithms of the binding constants. This relationship will be used for the prediction of the binding constants for CyD and other host-guest systems.
A planar amide bond is a fundamental linkage in the structures of peptides and proteins. The rigid planarity of the amide linkage, due to a conjugation between carbonyl and amine groups, may be requisite for encoded protein folding and many other biological processes. Non-planar amides in the ground state will decode the significance of the planarity and rigidity of the amide linkage. We show here that simple amides of 7-azabicyclo[2.2.1]heptane, free from steric bias, including parent N-benzoyl 7-azabicyclo[2.2.1]heptane, are nitrogen-pyramidal amides in the crystalline state. We can suggest that pyramidalized amide nitrogen is a general feature and intrinsic to the 7-azabicyclo[2.2.1]heptane motif. Low rotational barriers of the amide C-N bond in a series of N-benzoyl amides of 7-azabicyclo[2.2.1]heptane, compared to monocyclic amides, may imply that ground-state nitrogen pyramidalization of the former amides also exist in solution. The 7-azabicyclo[2.2.1]heptane motif also favors nitrogen pyramidalization of sulfonamides and N-nitrosoamines, which can lead to pharmacophores after appropriate modification.
To determine the prescription characteristics of β-agonist metered-dose inhalers (MDI), we retrospectively investigated all prescriptions containing one of five types of β-agonist MDIs available at Yamagata University Hospital in 1997, as well as patients’ characteristics. The total number of asthmatic patients was 225 (age, 11-79, mean, 47.2) in 1997. Fenoterol MDI was prescribed to patients who visited the hospital at regular periods and had more severe asthma. Isoprenaline MDI also was not prescribed for first-time patients. Patients who were prescribed tulobuterol MDI had mild or moderate asthma and some of them were only occasional or first-time visitors. Salbutamol and procaterol MDIs were also prescribed for first-time patients; however, tulobuterol MDI was the most frequently prescribed for first-time patients. Patients prescribed fenoterol and isoprenaline MDIs had adequate knowledge of proper asthma management, because sufficient information had been provided about the use of MDIs in the past. Patients prescribed tulobuterol MDI should be provided with detailed instructions because they had little knowledge of handling MDIs and self-management of asthma as many of them were first or intermittent visitors. Patients prescribed salbutamol or procaterol MDIs should be evaluated regarding their past medications and some of them should be instructed regarding the use of the MDI. Although these clinical aspects might be applicable only to our hospital, the same or other prescription patterns will be found in other hospitals and/or by other physicians. Adequate instructions to individual patients who are prescribed a particular β-agonist MDI should be provided by the medical staff, especially to outpatients, to reduce hospitalization and death from asthma.
We examined physiological and genetic factors affecting acetohexamide reductase (AHR) and 20β-hydroxysteroid dehydrogenase (20β-HSD) activities in liver microsomes of experimental animals. Pronounced strain-related differnces were found in both activities of AHR and 20β-HSD present in liver microsomes of male rats. Among rat strains tested in this study, even though a Wistar-Imamichi (WIM) rat strain was taken to lack AHR activity, it exhibited a significant 20β-HSD activity. These findings appeared to be in conflict with our conclusion reported so far, which AHR and 20β-HSD present in liver microsomes of male rats are identical enzymes. Thus the reason for this discrepancy was discussed. Furthermore, AHR and 20β-HSD activities were little or not observed in liver microsomes of female rats or male experimental animals other than the rat, indicating the existence of sex- and species-related differences in these two enzyme activities.
The effects of Selenium (Se) on lipid metabolism was studied in male Wistar rats which were fed a high-cholesterol diet (HCD) containing 1% (w/w) cholesterol and 0.5% (w/w) cholic acid for 10 weeks. Se was orally administered at daily doses of 0.173 mg/kg in HCD into the test rats for 10 weeks. As compared with control groups, Se/HCD suppressed the amounts of triglyceride (TG), total cholesterol (CH) and free fatty acid in the serum. Se/HCD also decreased the amounts of low-density lipoprotein cholesterol (LDL-C) in the serum. Further-more, Se/HCD inhibited the amount of liver TG and CH. The activity of fatty acid synthetase in the HCD fed group was higher than in the Se/HCD fed group. These results suggest that Se may have recuperative effects for hypercholesterolemia.
The contact sensitization of 11 simple coumarins was examined by subcutaneous sensitizing of guinea pigs, and the structure-activity relationship and cross-reactivity were investigated. Esculetin, 4-methylesculetin, and daphnetin were found to be strong sensitizers, and 4-hydroxy-coumarin to be a moderate sensitizer. Other simple coumarins tested hed a weak sensitivity to mild sensitizers. The results suggest that the introduction of hydroxy group, especially adjacent substitution at the 6,7, and 8 positions of the coumarin ring with two hydroxy groups, may play an important role in exhibiting the contact sensitization activity. The cross-reactivity was observed between esculetin and 4-methylesculetin, esculin or isoscoporetin, and also between daphnetin and 4-methylumbelliferone or umbelliferone, although there was no mutual cross-reactivity between esculetin and daphnetin. It is interesting to note that guinea pigs, which had a weak sensitivity to umbelliferone, showed a strong cross-reactivity to daphnetin, while those, which had a weak sensitivity to daphnetin, showed a weak cross-reactivity to umbelliferone. It is assumed that a skin-protein conjugation at 5 or 6 positions of the coumarin ring is important to elicit the cross-reactivity of esculetin or daphnetin groups.
Given the rapid progress of the prescribing/dispensing split, the ability of pharmacies to obtain and stock a small quantity of pharmaceuticals is seen as essential. Toho Yakuhin’s experience in “divided package sales” (wholesalers open an original package supplied by the manufacturer and sell only a part of the contents) shows that the number of orders and sales amount, and the number of pharmacies ordering in sales have remarkably increased. One or two kinds of pharmaceuticals were ordered in 73% cases, and one or two units in about 50%. This implies that “divided package sales” are utilized to obtain rarely prescribed drugs. On the other hand, five or more kinds of pharmaceuticals were ordered in 10% of cases, and ten or more units in 17%. “Divided package sales” were more used for low-price drugs. These indicated that “divided package sales” seemed to be looked upon by pharmacies as a means of obtaining a small quantity of pharmaceuticals, and seemed to be used not only in an emergency but also routinely. There are several problems for wholesalers in operating “divided package sales”, such as frequent delivery, delivery cost and information supply. Pharmacies should obtain a small quantity of pharmaceuticals by the routine delivery and should share the delivery cost. Pharmaceutical manufacturers’ cooperation in printing necessary information on each immediate container will be useful. Though there could be alternative ways of obtaining a small quantity of pharmaceuticals, all of them have the matter of delivery. We believe “divided package sales” will contribute to this situation.
We investigated the antioxidative effects of fluvastatin (FV or (±)-FV), each enantiomer ((+)-FV, (−)-FV) and its major metabolites on lipid peroxidation using rat and human liver microsomes. The extent of NADPH induced microsomal (Ms) lipid peroxidation was determined by thiobarbituric acid (TBA) assay. The antioxidative effect of each compound was shown as the percentage of inhibition on the formation of TBA reactive substance (TBARS) against vehicle control. The antioxidative effects of alpha-tocopherol (Toc), a potent antioxidative vitamin, probucol (PR), a potent antioxidative drug, pravastatin (PV) and simvastatin (SV), HMG-CoA reductase inhibitors, were also tested. The (±)-FV inhibit the formation of TBARS by 40 to 70% depending on Ms concentrations. The antioxidative effects of PR and TOC were comparable to those of FV. The inhibitory effects of PV and SV on the formation of TBARS were less potent than (±)-FV, PR and TOC. (+)-FV, (−)-FV, and (±)-FV inhibited the formation of TBARS by approximately 50% using rat hepatic microsomes. The antioxidative effects of (+)-FV was comparable to that of (−)-FV using human hepatic microsomes. These results indicated that the antioxidative effects of (+)-FV were comparable to those of (−)-FV, although the HMG-CoA reductase inhibitory activity of (+)-FV was 30-fold higher than that of (−)-FV.